Ligands for use in therapeutic compositions for the treatment of hemostasis disorders

ABSTRACT

The invention, in general, features a method of treatment and/or prevention of a thrombotic pathological condition, in a mammal, which includes administering to the mammal in need of such treatment a therapeutically effective amount of a composition including an antibody directed against the C1 domain of Factor VIII, which is a partially inhibitory antibody of Factor VIII.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/298,560 (pending), filed Dec. 9, 2005 (published as US 2006-0115474A1 on Jun. 1, 2006), which is a continuation-in-part of U.S. applicationSer. No. 10/030,522, filed May 2, 2002 (issued as U.S. Pat. No.7,067,313 on Jun. 27, 2006), which is a U.S. National Stage applicationof PCT/EP2000/06677, filed Jul. 13, 2000, which, in turn, claims thebenefit of U.S. Provisional Patent Application No. 60/143,891, filedJul. 14, 1999 and British Patent Application No. GB 9916450.1, filedJul. 14, 1999. application Ser. No. 11/298,560 is also acontinuation-in-part of International Patent ApplicationPCT/BE2004/000118, filed Aug. 16, 2004, which claims the benefit ofBritish Patent Application Nos. GB 0319118.6, filed Aug. 14, 2003 and GB0319345.5, filed Aug. 18, 2003. The entire contents of each of thesepatent and patent applications is hereby incorporated by reference inthis application.

FIELD OF THE INVENTION

The present invention relates to novel cell lines and to ligands, namelyhuman and/or humanized monoclonal antibodies, as well as fragments suchas Fab, Fab′, F(ab′)₂, scFv, single variable domains, complementarilydetermining regions, derivatives, homologs and combinations thereof,obtainable from the said cell lines. It also relates to pharmaceuticalcompositions comprising said ligands and to methods of preventing andtreating coagulation disorders and resulting thrombotic pathologicconditions in humans by administration of the said ligands to patientsin need thereof. It also relates to methods of obtaining specificmammalian antibodies.

BACKGROUND OF THE INVENTION

The formation of blood clots does not only limit bleeding in case of aninjury (hemostasis), but may lead to serious organ damage and death inthe context of atherosclerotic diseases by occlusion of an importantartery or vein. Thrombosis is thus blood clot formation at the wrongtime and place. It involves a cascade of complicated and regulatedbiochemical reactions between circulating blood proteins (coagulationfactors), blood cells (in particular platelets) and elements of aninjured vessel wall. Anticoagulation and antithrombotic treatment aim atinhibiting the formation of blood clots in order to prevent thesedangerous consequences, such as myocardial infarction, stroke, loss of alimb by peripheral artery disease or pulmonary embolism. Given theimportance of these diseases, it is rather surprising thatantithrombotic therapy relied on a few drugs for many years, namelyAspirin to inhibit platelets, Heparin that indirectly inhibits thecoagulation Factors IX, X and II (thrombin), and oral Warfarin thatinhibits Vit K-dependent factors (VII, IX, X, II and Prot C). After sometime, low molecular weight Heparins (inhibiting Factors X and II tovarious degrees) became anticoagulants of choice, largely because oftheir ease of application (once a day subcutaneous injection with nomonitoring need). With growing understanding of the processes involvedin thrombosis a growing number of specific inhibitors of coagulationfactors have been developed. However, a better efficacy/safety ratiocould to date not be obtained with them. Direct thrombin inhibitors, inparticular, were linked to increased bleeding complications in largeclinical trials.

Aspirin also provides a protective effect against thrombosis. It inducesa long-lasting functional defect in platelets, detectable clinically asa prolongation of the bleeding time, through inhibition of thecyclooxygenase activity of the human platelet enzyme prostaglandinH-synthase (PGHS-1) with doses as low as 30 to 75 mg. Sincegastrointestinal side effects of aspirin appear to be dose-dependent,and for secondary prevention, treatment with aspirin is recommended foran indefinite period, there are practical reasons to choose the lowesteffective dose. Further it has been speculated that a low dose (30 mgdaily) might be more anti-thrombotic but attempts to identify theoptimal dosage have yielded conflicting results. It has been claimedthat the dose of aspirin needed to suppress fully platelet aggregationmay be higher in patients with cerebrovascular disease than in healthysubjects and may vary from time to time in the same patient. However,aspirin in any daily dose of 30 mg or higher reduces the risk of majorvascular events by 20% at most, which leaves much room for improvement.

Further, the inhibiting role of aspirin may lead to prevention ofthrombosis as well as to excess bleeding. The balance between the twodepends critically on the absolute thrombotic versus hemorrhage risk ofthe patient.

In patients with acute myocardial infarction, reduction of infarct size,preservation of ventricular function and reduction in mortality has beendemonstrated with various thrombolytic agents. However these agentsstill have significant shortcomings, including the need for largetherapeutic doses, limited fibrin specificity, and significantassociated bleeding tendency. Recombinant tissue plasminogen activator(t-PA) restores complete patency in just over one half of patients,whereas streptokinase achieves this goal in less than one third.Further, reocclusion after thrombolytic therapy occurs in 5 to 10% ofcases during the hospital stay and in up to 30% within the first yearaccording to Verheugt et al., J. Am. Coll. Cardiol. (1996) 27:618-627.Thus numerous studies have examined the effects of adjunctiveantithrombin therapy for patients with acute myocardial infarction. Asan example, U.S. Pat. No. 5,589,173 discloses a method for dissolvingand preventing reformation of an occluding thrombus comprisingadministering a tissue factor protein antagonist, which may be amonoclonal or polyclonal antibody, in adjunction to a thrombolyticagent. Monoclonal antibodies have already been shown to be oftherapeutic value as antithrombotic agents. The first approved drug inthis field was Abciximab (ReoPro™), a humanized Fab fragment of a murinemonoclonal antibody (7E3) against platelet GP IIbIIIa receptors. Murineantibodies have characteristics which may severely limit their use inhuman therapy. As foreign proteins, they may elicit ananti-immunoglobulin response termed human anti-mouse antibody (HAMA)that reduces or destroys their therapeutic efficacy and/or provokesallergic or hypersensitivity reactions in patients, as taught by Jafferset al., Transplantation (1986) 41:572. The need for readministration intherapies of thromboembolic disorders increases the likelihood of suchimmune reactions. While the use of human monoclonal antibodies wouldaddress this limitation, it has proven difficult to generate largeamounts of such antibodies by conventional hybridoma technology.

Recombinant technology has therefore been used to construct “humanized”antibodies that maintain the high binding affinity of murine monoclonalantibodies but exhibit reduced immunogenicity in humans. In particular,chimeric antibodies have been suggested in which the variable region (V)of a non-human antibody is combined with the constant (C) region of ahuman antibody. As an example, the murine Fc fragment was removed from 7E3 and replaced by the human constant immunoglobulin G Fab region toform a chimera known as c7 E3 Fab or abciximab. Methods of obtainingsuch chimerical immunoglobulins are described in detail in U.S. Pat. No.5,770,198.

The potential for synergism between GPIIb/IIIa inhibition by monoclonalantibody 7 E3 Fab and thrombolytic therapy was evaluated by Kleiman etal., J. Am. Coll. Cardiol (1993) 22:381-389. Major bleeding was frequentin this study. Hence, the potential for life-threatening bleeding isclearly a major concern with this combination of powerful antithromboticcompounds.

Tissue Factor (TF), being a membrane glycoprotein functioning as areceptor for Factor VII and VIIa and thereby initiating the saidextrinsic pathway, has been investigated as a target for anticoagulanttherapy. In addition to this role, TF has been implicated in pathogenicconditions such as vascular disease and gram-negative septic shock. Astudy attempting to characterize the anticoagulant potential of murinemonoclonal antibodies showed that the inhibition of TF function by mostof the monoclonal antibodies assessed was dependent upon thedissociation of the TF/VIIa complex that is rapidly formed when TFcontacts plasma. One monoclonal antibody, TF8-5G9, was capable ofinhibiting the TF/VIIa complex without dissociation of the complex, thusproviding an immediate anticoagulant effect in plasma, as disclosed inWO 96/40,921.

Targeted clotting factors exhibit both a medium molecular weight range(about 45,000 to 160,000) and a relatively high normal plasmaconcentration (at least 0.01 micromol/L).

One persistent concern with all available anti-thrombotic agents is therisk of overdose and therefore of excessive and life-threateningbleeding. Most current antithrombotic agents therefore warrant closemonitoring of the patient.

Thus, there is a need for efficient compounds for the treatment ofcoagulation disorders, which cannot be overdosed, require no monitoringand are free from bleeding problems. For a therapeutic agent based onantibodies, the ideal compound would be a human antibody with fullanticoagulant efficacy that does not induce immunogenicity.

Factor VIII is a protein providing important_coagulant cofactor activityand is one of human clotting factors with a rather high molecular weight(265,000) and a very low normal plasma concentration (0.0007micromol./litre). With its 2,332 amino-acid residues, Factor VIII is oneof the longest known polypeptide chains and is synthesized in the liver,the spleen and the placenta. Its gene has been shown to include 186,000nucleotides.

Factor VIII circulates as inactive plasma protein. Factors V and VIIIare homologous proteins sharing a common structural configuration oftriplicated A domains and duplicated C domains with structurallydivergent B domains connecting the A2 and A3 domains. Factor VIIIcirculates in a multiplicity of fragmented species in a tightlyassociated complex with von Willebrand factor at a concentration of 1nmol/L. Factor VIII activation occurs by a cleavage between the A1 andA2 domains, resulting in the unstable heterotrimeric Factor VIIIamolecule. Factor VIIIa binds tightly to membranes that contain acidicphospholipids. Factor VIII contains a phospholipid binding site in theC2 domain, between amino-acids 2302 and 2332, according to Arai et al.in J. Clin. Invest. (1989) 83:1978. Within the same Factor VIII region,there is also a von Willebrand factor binding site acting in conjunctionwith amino-acid residues 1645-1689 in the A3 domain according to Shimaet al. in Throm. Haemost. (1993) 69:240 and J. Biol. Chem. (1994)269:11601.²

Polyclonal antibodies inhibiting the co-factor activity of Factor VIIIhave been classified as type I or type II inhibitors according to theircapacity to inhibit Factor VIII either completely (type I) or onlypartially (type II). According to Gawryl et al., Blood (1982) 60:1103-9,the reduced inactivation of Factor VIII by human type II autoantibodiesis believed to be due to a steric effect of von Willebrand factor.Monoclonal antibodies_are not mentioned and, to date, no therapeutic usewas made of such type II inhibitors. Biggs et al., Br. J. Haematol.(1972) 23:137 previously provided an interpretation derived from dataobtained by using human polyclonal antibodies, that a type II inhibitorypattern could be related to low affinity. B. Ly et al., ScandinavianJournal of Haematology (1982), 28:132-140 discloses polyclonalantibodies to Factor VIII which most often belong to the IgG class bothin hemophiliacs developing alloantibodies and in the more rare patientshaving autoantibodies against their own Factor VIII. These polyclonalantibodies partially inactivate Factor VIII activity like the antibodiesdescribed in Biggs et al. (1972) and Hoyer et al. (1982), This documentagain fails to mention whether monoclonal antibodies can reproduce thepattern of Factor VIII inactivation shown by patient's polyclonalantibodies. Again, no monoclonal antibodies are mentioned.

European patent applications EP-A-123,945, EP-A-152,746 and EP-A-432,134all disclose monoclonal antibodies produced by hybridoma cell lines andhaving a specific reactivity pattern with Factor VIIIc polypeptidefragments. These monoclonal antibodies are said to be useful fordetecting the presence of Factor VIIIc and related polypeptides inplasma by immunoassay techniques, but a therapeutic potential use is notsuggested in these documents.

J. Battle et al., Annals of Hematology (1997) 75:111-115, discloses apolyclonal alloantibody from a patient with severe von Willebranddisease showing, alike a rabbit polyclonal antibody against vonWillebrand factor, a partial inhibitory activity to plasma Factor VIII.These polyclonal anti-Factor VIII antibodies therefore inactivate FactorVIII following a pattern similar to anti-Factor VIII type II antibodiesfound in patients with hemophilia A (Gawryl et al., Blood (1982)60:1103-9). However, Factor VIII antibodies were not detected in thesaid human alloantibody, thus suggesting that it was a non- specificinhibition.

J. Ingerslev et al., Clinica Chimica Acta (1988) 174:65-82 discloses aseries of murine monoclonal antibodies against human von Willebrandfactor: two of them, belonging to the immunoglobulin isotype IgG1,exhibit an extremely low (1.3 BU/mg immunoglobulin) inhibition of FactorVIII as shown in table I of said document. By comparison, humanmonoclonal antibody BO2C11, derived from a hemophilia A patient withinhibitor, has a specific activity of 7,000 BU/ mg protein (Jacquemin etal. Blood, (1998) 92:496-506). This indicates that administration ofantibodies as described by Ingerslev to an animal or a human being wouldnot affect Factor VIII activity, unless an extremely high amount ofantibody (hundreds of mg/ml) was present in plasma. The authors do notdisclose whether when used in large excess these antibodies exhibitinhibitory activity like type I or type II (i.e. partial inactivation)polyclonal human Factor VIII inhibitor, such as described in Gawryl etal., Blood (1982) 60:1103-9.

Maraganore et al., Circulation (1992) 86:413, showed that a synthetic12-aminoacid peptide corresponding to residues 1675-1686 of Factor VIIIinhibits cleavage by thrombin of the heavy chain required for theactivation of the procoagulant activity of Factor VIII and also of thelight chain required to dissociate Factor VIII from von Willebrandfactor and that tyrosine sulfation of said peptide potentiates itsrecognition by Factor VIII.

O'Brien et al., J. Clin. Invest. (1988) 82:206-211 describes obtainingan animal model for hemophilia A by infusion of human anti-Factor VIIIantibody in rabbits. According to WO 95/01570, antibodies against thelight chain of human or porcine Factor VIIIc were produced in a firstanimal and subsequently a temporary hemophilia was induced in a secondanimal by means of the purified monospecific antibody obtained. U.S.Pat. No. 5,804,159 also discloses inducing a temporary clotting disorderin a mammal by means of an anti-plasma antibody preparation acting onseveral blood coagulation factors, e.g. a preparation comprisingantibodies against human von Willebrand factor and Factor VIII, oragainst Factor VIII/von Willebrand factor-complex, or againstprocoagulants, anticoagulants, clot structure factors, fibrinolysisfactors and phospholipids.

However, none of the above-mentioned antibodies compounds involvingFactor VIII have been described for therapeutic purposes. In fact thereis a prejudice among those skilled in the art against investigatinganti-Factor VIII antibodies for anti-thrombotic therapy because it isassumed that, a deficiency in Factor VIII being the cause of hemophiliaA, such antibodies would induce a bleeding state.

WO97/26010 discloses monoclonal antibodies having self-limitingneutralizing activity against a coagulation factor which are useful inpharmaceutical compositions for thrombotic disorders._Self-limitingneutralizing activity in this document is defined as the activity of anantibody that binds to a human coagulation factor and inhibitsthrombosis in a manner such that limited modulation of coagulation isproduced. Limited modulation of coagulation in turn is defined as anincrease in clotting time as measured by prolongation of the activatedpartial thromboplastin time (aPTT) where plasma remains clottable withaPTT reaching a maximal value, preferably 35 to 100 seconds, despiteincreasing concentrations of the monoclonal antibody. APTT is thus usedas the primary criterion for the evaluation of efficacy versus bleedingliability of antithrombotic agents.

More particularly, this document demonstrates that a sheep polyclonal toFactor VIII (SAF8C-IG, purchased from Affinity Biologicals) induces aself-limiting prolongation of aPTT (the aPTT increased to a maximum ofabout 65 seconds). We have demonstrated, however, that SAF8C-IG totallyinhibits the activity of human Factor VIII (see FIG. 10), i.e. is a typeI inhibitor in the classification of Gawryl et al., Blood (1982) 60:1103-9. This demonstrates that a limited increase in clotting time up toa certain maximum value is not necessarily correlated with partialinactivation of a clotting factor, and far less to a decrease in therisk of bleeding. For instance, it is well known that patients with acomplete deficit of coagulation factors have a limited prolongation ofaPTT, usually in the area of 60 to 100 seconds, but are neverthelessexposed to a dramatic risk of bleeding (Hathaway et al. Am J Clin Pathol(1979) 71: 22-25, and Hoffmann et al. Thromb Haemostas (1978) 39:640-645).

Conversely, it is well known that a prolonged APTT does not provide avalid parameter of the reduction of thrombosis risk. Notably, deficiencyin Factor XII, another coagulation factor of the intrinsic coagulationpathway results in APTT prolonged up to 6-fold (Hathaway et al. Am JClin Pathol (1979) 71: 22-25, and; Hoffmann et al. Thromb Haemostas(1978) 39: 640-645). However, a significant number of patients with thisdeficiency have experienced myocardial infarction or thromboembolism,demonstrating the lack of protection from thrombotic disease in patientdeficient in Factor XII, despite important prolongation of the APTT(McPherson R A Am J Clin Pathol (1977) 68: 420, and; Glueck H I et al.Ann Intern Med (1966) 64:390).

Jacquemin et al. in Blood (1998) 92:496-506 refers to a FactorVIII-specific human IgG4 monoclonal antibody (BO2C11) produced by a cellline derived from the memory B-cell repertoire of a hemophilia A patientwith inhibitors. BO2C11 is said to recognize the C2 domain of FactorVIII and to inhibit its binding to both von

Willebrand factor and phospholipids. It is said to completely inhibitthe procoagulant activity of native and activated Factor VIII with aspecific activity of 7,000 Bethesda units/mg. The present inventors havefurther shown that BO2C11, while totally inhibiting the activity ofhuman Factor VIII, provides a prolongation of about 110 seconds inclotting time as measured by aPTT, which again demonstrates that anincrease in clotting time up to a certain maximum value is notnecessarily correlated to partial inactivation of a coagulation factor.Such a reduction of Factor VIII levels would expose the patient tosevere risks of bleeding, like in patients with severe hemophilia A(Levine P H Ann NY Acad Sci (1975) 240:201; Gilbert M S Mount Sinai JMed (1977) 44: 339).

SUMMARY OF THE INVENTION

The present invention is related to new ligands, namely new monoclonalhuman or humanized antibodies, fragments, derivatives and homologsthereof, which bind to a factor involved in hemostasis, in particular toa factor or factors of the coagulation cascade and more in particularbind to Factor VIII or a complex thereof. The present invention furtherprovides polypeptides and other molecules which bind to a factor orfactors involved in hemostasis. The invention provides novel cell linesfrom which said monoclonal antibodies may be obtained. The inventionprovides pharmaceutical compositions comprising the ligands of theinvention and methods of prevention and treatment of coagulationdisorders and resulting thrombotic pathologic conditions in humans bythe administration of said ligands to patients in need thereof.

A first main object of the present invention is therefore to provide aneffective and safe anti-thrombotic therapy which reduces the risk ofbleeding in mammals, more particularly in humans.

It is a further object of this invention to provide therapeuticcompositions which provide an effective anti-thrombotic therapy whichreduces the risk of bleeding in mammals, more particularly in humans.

It is still a further object of the present invention to provide ananti-thrombotic therapy and anti-thrombotic therapeutic compounds whichare safer to use than the previously known therapies and compositions.

One aspect of the present invention is to target a human protein factorinvolved in hemostasis, in particular in the coagulation cascade, moreparticularly Factor VIII or a complex thereof, using specific inhibitoryligands. Preferably, these ligands, being other than polyclonalantibodies, provide a therapeutically useful plateau level of inhibitionby only partially inhibiting the function of the targeted factor so thata residual activity of the factor remains, even when the ligand is usedin a molar excess. A curve may be established of the inhibiting effectof a ligand in accordance with the present invention with respect to acertain targeted factor against the concentration of the said ligand andthe concentration may be determined at which a minimal residual factoractivity still exists which is at least 1%, preferably at least 2%. Theresidual factor activity at five times this concentration should not besubstantially different from the residual activity at the minimal point.

It is especially a further aspect of the present invention to providehigh affinity monoclonal antibodies, both human and humanized, as wellas fragments, derivatives, and homologs of any of these, having thecapacity to only partially inactivate a factor or factors in hemostasis,in particular in the coagulation cascade and more in particular FactorVIII or a complex thereof, even in molar excess of the ligand, therebypreventing the risk of overdosage and the resulting bleedingcomplications. In a particular embodiment, the present inventionprovides ligands, more in particular (high affinity and purified)monoclonal antibodies binding to the C1 domain of Factor VIII, bothhuman and humanized antibodies, as well as fragments, derivatives, andhomologs of any of these, having the capacity to only partiallyinactivate Factor VIII.

In a yet more particular embodiment, the invention provides monoclonalantibody Krix-1, obtained from the Krix-1 cell line as deposited withthe Belgian Coordinated Collections of micro-organisms, under accessionnumber LMBP 5089CB, as well as fragments, derivatives, and homologsthereof.

In one particular embodiment, the present invention provides monoclonalantibodies binding to the C1 domain of Factor VIII, most particularlyantibodies which compete with the binding of the antibody Krix-1produced by Krix-1 cell line deposited with the Belgian CoordinatedCollections of micro-organisms, under accession number LMBP 5089CB toFactor VIII as well as fragments, derivatives, and homologs thereof.Such competition for binding to Factor VIII can be tested with an ELISAas described herein.

In a further particular embodiment, the present invention providesmonoclonal antibodies and fragments thereof capable of binding to the C1domain of Factor VIII and capable of competing with the Krix-1 antibodyfor the binding to Factor VIII, more particularly binding to the sameantigen, most particularly to the same epitope as is bound by theantibody Krix-1.

In another particular embodiment, the present invention providesmonoclonal antibodies and fragments thereof binding to the C1 domain ofFactor VIII, which are derivatives, more particularly modified versionsof the Krix-1 antibody, comprising a variable heavy chain sequence beingat least 80% identical to the amino acid sequence depicted in FIG. 8(SEQ ID NO: 2) and/or a variable light chain sequence being at least 80%identical to the amino acid sequence depicted in FIG. 9 (SEQ ID NO:4).In a particular embodiment, the invention provides monoclonal antibodiesand fragments, derivatives and homologs thereof of which the variableheavy chain sequence, respectively light chain sequence, is al least90%, yet more in particular 95% identical to the amino acid sequencedepicted in FIG. 8 (SEQ ID NO: 2), and FIG. 9 (SEQ ID NO: 4),respectively. In another particular embodiment, said monoclonalantibodies binding to the C1 domain of Factor VIII, comprise a variableheavy chain sequence comprising at least one, more in particular twoCDRs being at least 80%, more in particular 90% or 95%, identical to theamino acid sequence of one of the CDRs depicted in FIG. 8 and/or avariable light chain sequence comprising at least one, more inparticular two, CDRs being at least 80%, more in particular 90% or 95%,identical to the amino acid sequence of the CDRs depicted in FIG. 9. Inanother particular embodiment, said monoclonal antibodies binding to theC1 domain of Factor VIII, comprise a variable heavy chain sequencecomprising CDRs being at least 80%, more in particular 90% or 95%,identical to the amino acid sequence of the corresponding CDRs depictedin FIG. 8 and/or a variable light chain sequence comprising CDRs beingat least 80%, more in particular 90% or 95%, identical to the amino acidsequence of the CDRs depicted in FIG. 9. In another particularembodiment, said monoclonal antibodies binding to the C1 domain ofFactor VIII or fragments thereof, comprise a sequence comprising one oremore CDR sequences corresponding to SEQ ID NO: 33, SEQ ID NO: 34 and/orSEQ ID NO: 35 (corresponding to the sequence of CDR1, CDR2 and CDR3,respectively of the heavy chain variable region of Krix-1) or a sequencewhich comprises one ore more CDR sequences corresponding to sequenceshaving at least 80%, more particularly 90%, most particularly at least95% sequence identity with SEQ ID NO: 33, SEQ ID NO: 34 and/or SEQ IDNO: 35, within the corresponding CDR sequence. Additionally oralternatively, the monoclonal antibodies and fragments thereof accordingto the present invention comprise a sequence comprising one or more CDRsequences corresponding to SEQ ID NO: 36, SEQ ID NO: 37 and/or SEQ IDNO: 38 (corresponding to the sequence of CDR1, CDR2 and CDR3,respectively of the light chain variable region of Krix-1) or a sequencewhich comprises one or more CDR sequences having at least 80%, moreparticularly 90%, most particularly at least 95% sequence identity withSEQ ID NO: 36, SEQ ID NO: 37 and/or SEQ ID NO: 38, within thecorresponding CDR sequence.

In a further particular embodiment, the invention provides derivativesof antibodies directed against the C1 domain of Factor VIII, moreparticularly derivatives of the antibodies described herein, such as butnot limited to derivatives of the Krix-1 antibody, which are modifiedantibodies or modified antibody fragments. Most particularly themodified antibody or antibody fragment is an antibody or fragment with amodified glycosylation, more specifically a modification of theglycosylation in the variable regions of the antibody or fragment, mostparticularly in one or more of the CDRs of the antibody or antibodyfragment, whereby the antibody or fragment still binds Factor VIII andpartially inactivates Factor VIII activity. A further particularembodiment of the present invention provides antibodies and fragmentswhich are modified versions of the Krix-1 antibody, which comprise amutated glycosylation site at one or more of positions Asn47 to Thr49 ofthe heavy chain variable region. Most particularly the inventionprovides antibodies and fragments which are modified versions of theKrix-1 antibody, whereby the modifications are selected from the groupconsisting of heavy chain variable region Asn47 changed to G1n47(KRIX-1Q), heavy chain variable region Asn47 changed to G1u47 (KRIX-1E)or heavy chain variable region Asn47 changed to Asp47 (KRIX-1D) and/orheavy chain variable region Thr49 changed to A1a49 (KRIX-1A).

In another embodiment, the invention provides monoclonal antibody RHD5,obtained from the cell line as deposited with the Belgian CoordinatedCollections of micro-organisms, under accession number LMBP 6165CB, aswell as fragments, derivatives, and homologs thereof RHD5 binds to theC1 domain of Factor VIII and only partially inhibits the activity ofFactor VIII, namely for 97-98%. The present invention thus relates tothe human antibody RHD5, fragments, derivatives and homologs thereof,which bind to Factor VIII, as well as relating to the novel cell linedeposited with the Belgian Coordinated Collections of micro-organisms,under accession number LMBP 6165CB, from which RHD5 may be obtained; theinvention further provides pharmaceutical compositions comprising saidantibody RHD5, fragments, derivatives and homologs thereof and tomethods of prevention and treatment of coagulation disorders andresulting thrombotic pathologic conditions in humans by theadministration of said RHD5 antibody, fragments, derivatives andhomologs thereof, to patients in need thereof.

In one particular embodiment, the present invention provides monoclonalantibodies binding to the C1 domain of Factor VIII, most particularlyantibodies which compete with the binding of the antibody RHD5 producedby RHD5 cell line deposited with the Belgian Coordinated Collections ofmicro-organisms, under accession number LMBP 6165CB to Factor VIII, aswell as fragments, derivatives, and homologs thereof Such competitionfor binding to Factor VIII can be tested with an ELISA as describedherein.

In a further particular embodiment, the monoclonal antibodies binding tothe C1 domain of Factor VIII of the present invention, are antibodieswhich bind to the same antigen, more in particular to the same epitope,bound by the antibody RHD5 deposited with the Belgian CoordinatedCollections of micro-organisms, under accession number LMBP 6165CB.

In a further particular embodiment, the present invention providesmonoclonal antibodies binding to the C1 domain of Factor VIII, andfragments thereof which are derivatives, more particularly modifiedversions of antibody RHD5, comprising a variable heavy chain sequencebeing at least 80%, more in particular 90% or 95%, identical to theamino acid sequence depicted in FIG. 14 (SEQ ID NO: 30) and/or avariable light chain sequence being at least 80%, more in particular 90%or 95%, identical to the amino acid sequence depicted in FIG. 14 (SEQ IDNO: 32). In another particular embodiment, said monoclonal antibodiesbinding to the C1 domain of Factor VIII or fragments thereof, comprise avariable heavy chain sequence comprising at least one, more inparticular two CDRs being at least 80%, more in particular 90% or 95%,identical to the amino acid sequence of one of the CDRs of the variableheavy chain depicted in FIG. 14 and/or a variable light chain sequencecomprising at least one, more in particular two, CDRs being at least80%, more in particular 90% or 95%, identical to the amino acid sequenceof the CDRs depicted in the variable light chain in FIG. 14. In anotherparticular embodiment, said monoclonal antibodies binding to the C1domain of Factor VIII and fragments thereof, comprise a variable heavychain sequence comprising three CDRs being at least 80%, more inparticular 90% or 95%, identical to the amino acid sequence of the threeCDRs depicted in the variable heavy chain sequence of RHD5 in FIG. 14and/or a variable light chain sequence comprising CDRs being at least80%, more in particular 90% or 95%, identical to the amino acid sequenceof the three CDRs depicted in the variable light chain sequence of RHD5in FIG. 14. In another particular embodiment, said monoclonal antibodiesbinding to the C1 domain of Factor VIII or fragments thereof, comprise asequence comprising one or more CDR sequences corresponding to SEQ IDNO: 39, SEQ ID NO: 40 and/or SEQ ID NO: 41 (corresponding to thesequence of CDR1, CDR2 and CDR3, respectively of the heavy chain ofRHDS) and/or a sequence which comprises one ore more CDR sequencescorresponding to sequences having at least 80%, more particularly 90%,most particularly at least 95% sequence identity with SEQ ID NO: 39, SEQID NO: 40 and/or SEQ ID NO: 41, within the corresponding CDR sequence.Additionally or alternatively, the monoclonal antibodies and fragmentsthereof according to the present invention comprise a sequencecomprising one ore more CDR sequences corresponding to SEQ ID NO: 42,SEQ ID NO: 43 and SEQ ID NO: 44 (corresponding to the sequence of CDR1,CDR2 and CDR3, respectively of the light chain of RHDS) or a sequencewhich comprises one or more CDR sequences having at least 80%, moreparticularly 90%, most particularly at least 95% sequence identity withSEQ ID NO: 42, SEQ ID NO: 43 and/or SEQ ID NO: 44, within eachcorresponding CDR sequence.

It is still another aspect of the present invention to provide novelcell lines producing the respective monoclonal antibodies disclosedherein. A particular embodiment of the present invention provides celllines of human monoclonal antibodies capable of binding to the C1 domainof Factor VIII and capable of partially inhibiting Factor VIII activity.A further particular embodiment of this aspect of the present inventionprovides the Krix-1 cell line as deposited with the Belgian CoordinatedCollections of micro-organisms, under accession number LMBP 5089CB andthe RHDS cell line, deposited with the Belgian Coordinated Collectionsof micro-organisms, under accession number LMBP 6165CB.

Yet a further aspect of the present invention provides polynucleotidesequences which encode the antibodies or fragments thereof mentionedabove. It will be appreciated that a multitude of nucleotide sequencesexist which fall under the scope of the present invention as a result ofthe redundancy in the genetic code. The present invention also includescomplementary sequences to the sequences encoding the monoclonalantibodies, or fragments thereof, mentioned above. In particular, thepresent invention includes probes constructed from the monoclonalantibodies, or fragments thereof, mentioned above or from thepolynucleotides or from the complementary sequences mentioned above.Particular embodiments of the nucleotide sequences provided inaccordance with the present invention include the nucleotide sequencesencoding the heavy and light chain CDRs of Krix-1, i.e. corresponding tothe nucleotide sequences encoding the amino acid sequences provided inSEQ ID Nos 33, 34, 35, 36, 37, and 38. Further particular embodiments ofthe nucleotide sequences provided in accordance with the presentinvention include the nucleotide sequences encoding the heavy and lightchain CDRs of RHDS, i.e. corresponding to the nucleotide sequencesencoding the amino acid sequences provided in SEQ ID Nos 39, 40, 41, 42,43, and 44. Further embodiments include nucleotide sequences encodingmodified versions of the above-mentioned CDRs, which encode a proteincapable of binding to Factor VIII and of partially inhibiting FactorVIII activity. More particular embodiments of the present inventioninclude sequences encoding modified versions of the above-mentionedCDRs, which modified versions comprise modifications resulting in themodified glycosylation of the CDRs, more particularly the modifiedN-glycosylation of the CDRs. Most particular embodiments of thenucleotide sequences of the present invention include sequences encodinga modified version of the Krix-1 antibody or a fragment thereof whichcomprises a mutated glycosylation site at one or more of positions Asn47to Thr49 of the heavy chain variable region, most particularly sequencesencoding antibodies and fragments thereof which are modified versions ofthe Krix-1 antibody, whereby the modifications are selected from thegroup consisting of heavy chain variable region Asn47 changed to G1n47(KRIX-1Q), heavy chain variable region Asn47 changed to G1u47 (KRIX-1E)or heavy chain variable region Asn47 changed to Asp47 (KRIX-1D) and/orheavy chain variable region Thr49 changed to A1a49 (KRIX-1A).

Yet a another aspect of the present invention provides a method ofattenuation of coagulation in humans, comprising administering a ligand,being other than a polyclonal antibody, such as a monoclonal antibody,either human or humanized, a fragment, derivative or homolog thereof,capable of only partially inactivating a factor or factors inhemostasis, in particular in the coagulation cascade and more inparticular Factor VIII or a complex thereof to a patient in need of suchattenuation even when the said ligand is in a molar excess. It furtherprovides a method of treatment or prevention of a thrombotic pathologiccondition in mammals, namely in humans, comprising administering atherapeutically effective amount of a ligand, other than a polyclonalantibody, for instance a monoclonal antibody, either human or humanized,or a fragment, derivative or homolog thereof, capable of only partiallyinactivating, even when the said ligand is in a molar excess, a factoror factors involved in hemostasis, in particular in the coagulationcascade, and more particularly Factor VIII or a complex including FactorVIII, to a mammal in need of such treatment or prevention. In apreferred embodiment, the thrombotic pathologic condition may beselected for instance from intravascular coagulation, arterialthrombosis, arterial restenosis, venous thrombosis and arteriosclerosis.The methods of treatment and prevention of a thrombotic pathologicalcondition in a mammal of the present invention comprise theadministration of one or more of the ligands of the present invention,capable of partially inhibiting Factor VIII, to a mammal in needthereof.

Another aspect of the present invention is directed to providing acomposition, more in particular a pharmaceutical composition comprisinga ligand, other than a polyclonal antibody, having the capacity ofbinding to a site on a factor or factors involved in hemostasis, inparticular in the coagulation cascade, and more particularly Factor VIIIor a complex including Factor VIII, for only partially inactivating thesaid factor or factor complex even when the ligand is in molar excess,in admixture with a pharmaceutically acceptable carrier. The said ligandpreferably is a high affinity anti-Factor VIII or anti-Factor VIII—vonWillebrand factor complex monoclonal antibody, either human orhumanized, or hybridized, or a fragment, derivative or homolog thereofParticular embodiments of the pharmaceutical compositions providedcomprise one or more of the ligands of the present invention describedherein, capable of partially inhibiting Factor VIII. The pharmaceuticalcomposition of the present invention may further optionally comprise atherapeutically effective amount of a thrombolytic agent.

Another aspect of the present invention is directed to providing methodsfor the selection of specific monoclonal antibodies. The conventionaltechnique of immunizing an animal such as a mouse with a protein such asFactor VIII elicits an immunological response which may involve severalepitopes on the Factor VIII molecule. The present invention providesmore selective methods of obtaining specific monoclonal antibodiesagainst an epitope of a wild-type protein. First, a donor, e.g. a mammalsuch as a human, is provided (i.e. selected) which has an at leastpartially functional modified version of a wild-type protein. Themodification, which more particularly lies in a domain of the protein ofinterest, may be due to any cause, e.g. race or variety, to geneticdefects at birth, to an illness or by human interference, e.g.immunotolerance against the functionally modified version. The mammaldonor is then administered the wild-type protein in order to elicit animmune response; at this stage, it is important that a sufficientquantity of the wild-type protein (e.g. Factor VIII) be administereduntil an immune response is generated. Then, in a final step of themethod, selection of B-cells from the mammal donor will result in a muchgreater chance of obtaining monoclonal antibodies against an epitope inthe region of the modification, for instance by selecting B-lymphocytesfrom the donor which produce antibodies only partially inactivating thewild type protein.

The anticoagulant potential of inhibiting Factor VIII has to date notbeen explored, perhaps because of the well known bleeding complicationsthat occur in hemophilia A patients that lack Factor VIII activitycompletely (severe hemophilia) or to a large extent (moderatehemophilia). Hemophilia A, however, not only demonstrates the importanceof Factor VIII as limiting co-factor of coagulation, but also theexisting link between coagulation and the development ofatherosclerosis. Atherosclerosis and its thrombotic complications wereindeed found to be significantly rarer among patients with hemophilia A.Antagonizing Factor VIII activity at a level that allows sufficienthemostasis to prevent bleeding but protects from pathologicintravascular thrombus formation therefore holds substantial promise forsafe anticoagulation in prothrombotic diseases such as deep veinthrombosis (DVT), pulmonary embolism (PE), postoperatively, inpregnancy, in coronary artery disease (CAD), cerebrovascular disease(CVD), peripheral artery disease (PAD) and in vascular interventions.

The present invention is based on the surprising determination of newligands, namely new human and humanized monoclonal antibodies andfragments, derivatives and homologs thereof These may exhibit anunforeseen “plateau effect”, i.e. the achievement of only a partialinactivation of a factor involved in hemostasis, in particular in thecoagulation cascade, either individually or in combination, whatever theexcess of ligand. The ligands may bind to a factor or a complex offactors resulting in only partially impairing the function of aphysiologically functional site of the said factor or factor complex.This “plateau effect” makes the ligands particularly suitable fortreating coagulation disorders and resulting thrombotic pathologicconditions while minimizing the risk of bleeding, by comparison,notably, to antibodies with self-limiting neutralizing activitymentioned in WO97/26010, There is therefore a sharp contrast between the“the self-limiting neutralizing activity of antibodies to coagulationfactors disclosed in WO97/26010 and the clinically meaningful plateauinhibition that the present invention covers, where anti-Factor VIIIantibodies such as KRIX-1 inhibit

Factor VIII activity by no more than 85% or such as RHD5 inhibit FactorVIII activity by no more than 97-98%.

Particularly useful is a property of ligands in accordance with thepresent invention to allow some physiological function of the affectedtarget protein even when the ligand is in molar excess. The ligands maybe anti-Factor VIII antibodies or antibodies against a Factor VIIIcomplex, in particular human or human hybrid monoclonal antibodies whichbind to Factor VIII or a Factor VIII complex and at least partiallyinhibit the activity of Factor VIII. Data indicate that type IIinhibitors react with different antigenic determinants than type Iantibodies and that these determinants are partially blocked in theFactor VIII/von Willebrand factor complex.

The present invention will now be described in more details withreference to the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents the results of producing human monoclonal antibodiesderived from a hemophilia A patient, expressed in the form of IgGantibody binding to Factor VIII in ELISA.

FIG. 2 shows inhibition of Factor VIII activity by the monoclonalantibody BO2C11.

FIG. 3 shows inhibition of Factor VIII activity by the monoclonalantibody produced from the cell line KRIX-1.

FIG. 4 shows inhibition of the binding of activated Factor VIII onphosphatidyl-L-serine by the monoclonal antibody produced from the cellline KRIX-1.

FIG. 5 shows the influence of certain polyclonal antibodies on thedissociation of activated Factor VIII from von Willebrand factor.

FIGS. 6 and 8 show amino acid sequences (the lower lines) and nucleotidesequences (upper lines) for the variable regions V_(H) of the heavychains of BO2C11 and the KRIX-1 monoclonal antibodies, respectively.Also shown are the three complementarity determining regions (CDR) ofeach chain which are each an individual polypeptide ligand in accordancewith an individual embodiment of the present invention. (For FIG. 8: Asnand Thr residues of the glycosylation consensus site are indicated withan asterisk).

FIGS. 7 and 9 show amino acid sequences (the lower lines) and nucleotidesequences (upper lines) for the variable regions V_(L) of the lightchains of BO2C11 and the KRIX-1 monoclonal antibodies, respectively.Also shown are the three CDR's of each chain each of which is anindividual polypeptide ligand in accordance with particular embodimentsof the present invention.

FIG. 10 provides a graph showing inhibition of Factor VIII activity bythe antibody SAF8C-Ig mentioned in WO97/26010

FIG. 11 illustrates the kinetics of Factor VIII inhibition by KRIX-1.

FIG. 12 illustrates the inhibition of venous thrombosis in a hamstermodel by KRIX-1.

FIG. 13: Graph of experimental results showing the inhibition of FactorVIII binding to RHD5 by Krix-1 and RHD5. Biotinylated recombinant FactorVIII was mixed with different concentrations of RHD5 (closed symbols) orKrix-1 (open symbols) before addition to RHD5 coated plates. The plateswere then incubated for 2 hours at 4° C. and the binding of Factor VIIIwas detected by the addition of avidine peroxidase and OPD.

FIG. 14: nucleotide and amino acid sequence of RHD5 variable heavy andlight chain (Asn and Thr residues of putative glycosylation consensussites are indicated with an asterisk)

FIG. 15: Inhibition of Factor VIII functional activity in coagulationassays.

Equal volumes of RHD5 and of a pool of normal plasma were incubated for2 hours at 37° C. RHD5 concentrations before mixing with plasma were asindicated. The residual Factor VIII activity was measured in achromogenic Factor VIII assay. Results were expressed as the percentageFactor VIII inhibition in presence of RHD5 by comparison with samplestreated identically in the absence of RHD5.

FIG. 16: RHD5 inhibition of Factor VIII binding to vWF. Recombinantbiotinylated Factor VIII was mixed with different concentrations of RHD5before addition to vWF coated plates. The plates were then incubated for2 hours at room temperature and the binding of Factor VIII was detectedby the addition of avidine peroxidase.

FIG. 17: Epitope mapping of Krix-1 and RHD5 via immunoprecipitation.

FIG. 18: Graph of experimental results showing the inhibitory activityof native and deglycosylated KRIX-1, in accordance with an embodiment ofthe invention. KRIX-1 was deglycosylated by treatment withN-glycosidase-F. To assess the inhibitory activity of native (NAT;closed symbol) and deglycosylated KRIX-1 (DEG; open symbol), one volumeof antibody at various dilutions was mixed with one volume of a pool ofnormal human plasma and incubated for 2 h at 37° C. The residual FactorVIII activity was then measured in a chromogenic assay.

FIG. 19: Graph of experimental results showing that mixingdeglycosylated KRIX-1 with native KRIX-1 reduces the maximal “plateau”inhibition of Factor VIII, in accordance with an embodiment of theinvention. Normal plasma was incubated for 2 h at 37° C. with variousconcentrations of Krix-1, deglycosylated Krix-1, and mixtures of nativeand deglycosylated Krix-1 at a ratio of 4.5 and 1.5 native versusdeglycosylated antibody. After a 2h incubation period at 37° C., theresidual Factor VIII activity was measured in a Factor VIII chromogenicassay.

FIG. 20: Graph of experimental results showing the inhibitory activityof CHO-recKRIX-1 and KRIX-1 on Factor VIII activity in plasma, inaccordance with an embodiment of the invention. To assess the inhibitoryactivity of the antibody produced by the human cell line (KRIX-1) andthe recombinant antibody produced in CHO (CHO-recKRIX-1), one volume ofantibody at various dilutions was mixed with one volume of a pool ofnormal human plasma and incubated for 2 h at 37° C. The residual FactorVIII activity was then measured in a chromogenic assay.

FIG. 21: Graph of experimental results showing the effect of KRIX-1 andCHO-recKRIX-1 on vena cava thrombosis in mice, in accordance with anembodiment of the invention. Thrombus was induced in the inferior venacava 16 hours after subcutaneous administration of 150 microgram KRIX-1and CHO-recKRIX-1 or saline. Animals were sacrificed after 4 hours. Fivetransverse segments at 0.5 mm intervals through the infrarenal vena cavawere scored 1 if thrombus was present or zero if absent, and the scoreswere summed.

FIG. 22: Graph of experimental results showing that KRIX-1,CHO-rec-KRIX-1 protect against penile thrombosis and priapism in matedAT^(m/m) males, in accordance with an embodiment of the invention. Maleswere injected twice subcutaneously with vehicle (PBS), or with 100microgram antibody mAb Krix-1 or rec-mAB Krix-1, three days before andon the day of mating. Thrombotic outcome was scored zero if the micewere free of thrombosis at the end of the 8-day follow-up, 1 ifmicroscopic thrombosis without priapism was observed, 2 if macroscopicthrombosis without priapism occurred, and 3 if the males developedsevere thrombosis with irreversible priapism. (#) One mouse each in themAb Krix-1 or rec-mAb Krix-1 treated group was free of macroscopicthrombosis at the end of the experiment but could not be analyzed bymicroscopy and were therefore scored 1.

FIG. 23: Graph of experimental results showing the inhibitory activityof CHO-recKRIX-1 and mutated antibodies with N-glycosylation site in thevariable region, in accordance with an embodiment of the invention. Toassess the inhibitory activity of the antibodies, one volume of antibodyat various dilutions was mixed with one volume of a pool of normal humanplasma and incubated for 2 h at 37° C. The residual Factor VIII activitywas then measured in a chromogenic assay.

FIG. 24: Graph of experimental results showing the inhibitory activityof CHO-recKRIX-1 and CHO-recKRIX-1Q, in accordance with an embodiment ofthe invention. To assess the inhibitory activity of the antibodies, onevolume of antibody at various dilutions was mixed with one volume of apool of normal human plasma and incubated for 2h at 37° C. The residualFactor VIII activity was then measured in a chromogenic assay.

FIG. 25: Drawing representing the experimental protocol forextracorporeal thrombosis in baboons. Arterial and venous thrombogenicdevices. Arteriovenous shunts were implanted in male baboon femoralvessels. Thrombogenic devices prefilled with saline were incorporated asextension segments into the permanent arteriovenous shunt.Platelet-dependent arterial thrombus was induced by inserting Dacroninto the wall of Silastic tubing. Coagulation-dependent venousthrombosis was generated in an expansion chamber. The deposition ofautologous radiolabeled platelets was followed with a gammascintillation camera.

FIG. 26: A graph of experimental results showing the inhibition ofplatelet deposition in the arterial and venous thrombosis chambersbefore and after administration of CHO-recKRIX-1Q, in accordance with anembodiment of the invention. Platelet deposition was recorded as afunction of time in the expansion (”venous“) thrombosis chamber (A) andin the Dacron (”arterial“) thrombosis chamber (B) incorporated in anextracorporeal arteriovenous shunt implanted between femoral vessels. Inthe control studies, the devices were kept in place for 60 min or untilocclusion of the catheter. The baboons were then treated with a singleintravenous bolus of antibody. New thrombogenic devices were placed thenfor 60 minutes, 1 h, 24 h after the bolus injection. The extracorporealshunts were then removed.

FIG. 27: Graph of experimental results showing that CHO-recKRIX-1Qprotects against penile thrombosis and priapism in mated AT^(m/m) males,in accordance with an embodiment of the invention. Males were injectedtwice subcutaneously with vehicle (PBS), or with 100 μg antibodyCHO-recKRIX-1Q or a control IgG4 human monoclonal antibody (IgG4), threedays before and on the day of mating. Thrombotic outcome was scored zeroif the mice were free of thrombosis at the end of the 8-day follow-up, 1if microscopic thrombosis without priapism was observed, 2 ifmacroscopic thrombosis without priapism occurred, and 3 if the malesdeveloped severe thrombosis with irreversible priapism. (#) Animals freeof macroscopic thrombosis at the end of the experiment but which couldnot be analyzed by microscopy were scored 1.

FIG. 28: Graph of experimental results showing the inhibitory activityof native and deglycosylated Fab fragment of LCL-KRIX-1 and CHO-KRIX-1,in accordance with an embodiment of the invention. KRIX-1 wasdeglycosylated by treatment with N-glycosidase-F and Fab were producedby digestion with papain. To assess the inhibitory activity of intactantibodies and native and deglycosylated Fab, one volume of antibody atvarious dilutions was mixed with one volume of a pool of normal humanplasma and incubated for 2 h at 37° C. The residual Factor VIII activitywas then measured in a chromogenic assay.

FIG. 29: Graph of experimental results showing the Factor VIIIinhibitory activity of scFv fragment of KRIX-1 (scFv-KRIX-1VLVH(His))produced in Pichia pastoris, in accordance with an embodiment of theinvention. To assess the inhibitory activity of scFv-KRIX-1VLVH(His),one volume of buffer with scFvKRIX-1VLVH(His) at various concentrationswas mixed with one volume of a pool of normal human plasma and incubatedfor 2 h at 37° C. The residual Factor VIII activity was then measured ina chromogenic assay.

FIG. 30: Graph of experimental results showing the Factor VIIIinhibitory activity of scFv fragment of KRIX-1 and KRIX-1Q, inaccordance with an embodiment of the invention. To assess the inhibitoryactivity of scFv fragment of KRIX-1 and KRIX-1Q, one volume of culturesupernatant of CHO cells, transfected with an expression vector forscFv-KRIX-1VLVH(His) (open symbols) or scFv-KRIX-1VLVHQ(His) (closedsymbols), at various dilutions was mixed with one volume of a pool ofnormal human plasma and incubated for 2 h at 37° C. The residual FactorVIII activity was then measured in a chromogenic assay.

DEFINITIONS

The term “antibody” generally refers to an antibody of any origin suchas from human, murine, camel or other origin, and includes an antibodyof any class or isotype such as IgG, IgA, IgM, IgD, and IgE, and anysubclass within such a class, such as for IgG, IgG1, IgG2, IgG3 andIgG4.

The term “fragment” when referring to an antibody against Factor VIIIincludes molecules comprising either parts of both heavy and lightchains, (such as Fab, F(ab)₂, F(ab′)₂ or ScFV) or single heavy or lightchains (e.g. light chain dimers), optionally including their constantregion (or parts thereof), or optionally minor modifications (such asallotypic variants) of that constant region. It moreover includes partsof said heavy and/or light chains, such as the variable regions of theantibodies, subparts thereof, in particular the hypervariable (HV) orcomplementarity determining region(s) (CDR(s)). Thus antibody fragmentsinclude peptides made up of stretches of amino acids comprising at leastone CDR, optionally with adjacent framework sequences, e.g. of up toabout 10 amino acid sequences at one or both ends of the CDR(s).

A “complementarity determining region (CDR)” in the present inventionrefers to a hypervariable amino acid sequence isolated from or presentwithin an antibody variable region, which interacts with the epitope onthe antigen. Traditionally, based on their position in the intactantibody, CDR regions are numbered “CDR1”, “CDR2” and “CDR3” of thevariable light (VL) and heavy (VH) chains, respectively (also referredto as L1, L2, L3 and H1, H2, H3 respectively).

A “humanized” antibody or antibody fragment as used herein, refers toantibody molecules or fragments thereof in which amino acids have beenreplaced in the non-antigen binding regions in order to more closelyresemble a human antibody.

A “Reshaped” human antibody or antibody fragment or a “Human hybrid”antibody or antibody fragment as used herein, refers to a human antibodyor fragment thereof in which amino acids in the antigen binding regionshave been replaced with sequences in accordance with the presentinvention, e.g. CDR's, or other parts of variable regions which havebeen derived from the repertoire of human antibodies.

The term “native antibody” as used herein refers to the originalantibody as obtained from a cell line producing said antibody understandard culturing of a lymphoblastoid cell line, i.e. unmodified by theaddition of enzymes or by mutations. Such a native antibody is alsoreferred to as a wild-type antibody. For instance, in the context of thepresent invention, when reference is made to the native Krix-1 antibody,a comparison to the antibody as obtained from the Krix-1 cell line(deposited as LMBP 5089CB), under standard cultivation conditions isintended.

The term “derivative” of an antibody or antibody fragment as used hereinrefers to an antibody or fragment thereof which has been alteredchemically or genetically thereby retaining at least part of orimproving its ability to bind to the epitope of the native antibody.Examples of derivatives of antibodies and antibody fragments includeantibodies or fragments in which either the amino acid sequence has beenmodified and/or antibodies or fragments in which glycosylation has beenmodified as well as “polymorphisms” of antibodies.

An antibody or fragment having a modified amino acid sequence includes amolecule in which one or more amino-acids have been either substitutedby any other amino-acid residue or deleted compared to the nativeantibody. Such amino-acid substitution or deletion can be locatedanywhere in the antibody or antibody fragment molecule. It also includesmolecules in which amino-acid residues have been substituted and/ordeleted at more than a single location.

When referring in the present application to an antibody or antibodyfragment of which the glycosylation has been “modified” it is intendedto refer to antibodies or fragments thereof which have been engineeredor produced in a way that their glycosylation differs from that of thenative antibody, meaning that certain extra carbohydrates are present orcertain carbohydrates are missing, or both, relative to the nativeantibody. The modification of the glycosylation can occur at one or atdifferent positions in the antibody or antibody fragment.

The term “polymorphisms” refers to the result of the regular andsimultaneous occurrence in a single interbreeding population of two ormore alleles of a gene, where the frequency of the rarer alleles isgreater, typically greater than 1%, than can be explained by recurrentmutation alone.

The term “homology” or “homologous” as used herein with reference to theantibodies or antibody fragments in accordance with the presentinvention refers to a molecule which will compete with or inhibitbinding of one of the antibodies or antibody fragments in accordancewith the present invention to the antigen. The binding should bespecific, i.e. the binding of the alternative molecule should be asspecific to the antigen as the antibody or antibody fragment inaccordance with the present invention. Where used to refer to theantibodies or antibody fragments in accordance with the presentinvention, homology includes, but is not limited to, molecules having atleast 80%, more preferably 90% and most preferably 95% amino acidsequence similarity or sequence identity with the sequence of therelevant antibody or antibody fragment.

Sequence comparisons: Comparisons of protein or nucleotide sequences canbe designated in terms of sequence identity or sequence similarity.Nucleotide or amino acid sequences which are “identical” means that whentwo sequences are aligned, the percent sequence identity, i.e. thenumber of positions with identical nucleotides or amino acids divided bythe number of nucleotides or amino acids in the shorter of thesequences, is higher than 80%, preferably at least 90%, even morepreferably at least 95%, most preferably at least 99%, more specificallyis 100%. The alignment of two nucleotide sequences is performed by thealgorithm as described by Wilbur and Lipmann (1983) Proc. Natl. Acad.Sci. U.S.A. 80:726, using a window size of 20 nucleotides, a word lengthof 4 nucleotides, and a gap penalty of 4.

Two amino acids are considered as “similar” if they belong to one of thefollowing groups GASTCP; VILM; YWF; DEQN; KHR. Thus, sequences which aresimilar means that when the two protein sequences are aligned the numberof positions with identical or similar nucleotides or amino acidsdivided by the number of nucleotides or amino acids in the shorter ofthe sequences, is higher than 80%, preferably at least 90%, even morepreferably at least 95% and most preferably at least 99%, morespecifically is 100%.

Alternatively, comparisons of protein or nucleotide sequences can bedesignated in terms of number of the number of amino acids ornucleotides (or codons) that are different. When referring to a modifiedKrix-1 antibody for instance, it includes antibodies or antibodyfragments comprising an amino acid sequence in which the CDR sequences,when compared to native Krix-1 each differ in maximally 3 amino acids.The maximal number of modified amino acids within the CDRs, whencompared to Krix-1 is thus 18.

An “inhibitory antibody” or an “antibody with inhibitory activity” asused herein refers to an antibody which inhibits the activity of itstarget protein at least partially. The term “partial inhibition” or“plateau inhibition” as used herein refers to an inhibition of activitywhich is less than 100%.

The term “thrombotic pathological condition” or a thrombotic disorder isa disorder wherein unwanted clot or ‘thrombus’ is produced or whereinthe risk of clot formation is increased. Examples of thromboticpathological conditions include, but are not limited to intravascularcoagulation, arterial thrombosis, peripheral artery disease (PAD),coronary arterial disease (CAD), arterial restenosis, venous thrombosis(notably deep vein thrombosis (DVT), pulmonary embolism (PE), cerebralischemic disorders, atrial fibrillation and arteriosclerosis.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, reagents or componentsas referred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e. g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i. e., be embedded in a larger nucleic acid orprotein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be described with reference to certainembodiments and to certain figures but the present invention is notlimited thereto. In particular, the present invention will mainly bedescribed with reference to ligands to Factor VIII but the presentinvention is not limited thereto.

The present invention relates to a general concept of obtaining atherapeutically useful “plateau inhibition” of coagulation by onlypartially inactivating a factor involved in coagulation. Moreparticularly, the invention relates to the selection of certainmonoclonal antibodies, as well as to the production of such human orhumanized monoclonal antibodies, or fragments, derivatives or homologsthereof, and using these for anti-thrombotic therapy and inanti-thrombotic therapeutic compositions. These partial inhibitors andcompositions comprising such inhibitors have the advantageous propertythat the inactivation of the factor they target is only partial evenwhen the ligand is in a molar excess. This is of interest for thecoagulation cascade in that complete inhibition may cause side effectssuch as uncontrolled bleeding. Partial inhibition means that even thoughthe ligand is used in an amount which might be expected to inactivatecompletely the targeted factor, the inactivation is still incomplete,and that there is as such no risk of ‘overdosing’.

According to a first aspect, the present invention relates toantibodies, also referred to herein as ‘ligands’ herein, which arereactive with human Factor VIII and more specifically have the capacityof inactivating the co-factor activity of human factor VIII byinterfering with proteolytic cleavage site or von Willebrand factor ortenase complex reaction or by inducing a three-dimensionalconformational change in Factor VIII, in particular by targeting adomain of Factor VIII and by recognizing epitopes located in the saiddomain.

Thus, according to the invention, monoclonal antibodies are providedwhich inhibit Factor VIII activity. According to a particular embodimentFactor VIII activity refers to the ability of Factor VIII to act asco-factor in the coagulation cascade, which can be evaluated by itseffect on coagulation, such as but not limited to by the methodsdescribed herein (see below). This inhibition can be caused by the factthat Factor VIII is inhibited from binding other factors such as vWFand/or phospholipids. According to a particular embodiment of theinvention, the inhibitory antibodies are directed to the C1 domain ofFactor VIII. Although the inventors do not wish to be bound to a singleexplanation or theory, it is believed that the binding of such humanmonoclonal antibodies results in partial impairment of the binding ofactivated Factor VIII to phospholipids, a necessary step for cofactoractivity expression. According to a further particular embodiment, theantibodies of the present invention are of the class IgG.

A particular embodiment of the present invention relates to the KRIX-1antibodies produced by the KRIX-1 cell line described herein which hasbeen obtained as described herein. Krix-1 antibodies are directedagainst the C1 domain of Factor VIII and are capable of partiallyinhibiting the co-factor activity of Factor VIII. The invention furtherprovides antibodies capable of binding to the same antigen as the Krix-1antibody, more in particular antibodies binding to the same epitope asbound by the Krix-1 antibody, bound by the antibody produced by celllines mentioned hereinabove; even more particularly, the presentinvention provides antibodies which compete with Krix-1 for binding toFactor VIII as tested with an ELISA as described herein. Particularly,the antibodies have at least 80% sequence identity, more particularly atleast 90% sequence identity, even more particularly at least 95%sequence identity, most particularly at least 98% sequence identity,with the Krix-1 antibody within their CDR regions. Additionally oralternatively, the antibodies have an amino acid sequence which differsfrom Krix-1 in maximally 3 amino acids within each CDR. Moreparticularly, the total number of modified amino acids within all of theCDRs is 15, even more particularly the maximum number of modified aminoacids for all of the CDRs, compared to native Krix-1, is 12.

According to an alternative embodiment, the present invention relates toRHDS antibodies produced by the RHDS cell line described herein whichhas been obtained as described herein. RHDS antibodies are directedagainst the C1 domain of Factor VIII and are capable of partiallyinhibiting the co-factor activity of Factor VIII. The invention furtherprovides antibodies capable of binding to the same antigen as the RHDSantibody, more in particular antibodies binding to the same epitope asbound by the RHDS antibody, bound by the antibody produced by cell linesmentioned hereinabove; even more particularly, the present inventionprovides antibodies which compete with RHDS for binding to Factor VIIIas tested with an ELISA as described herein. Particularly, theantibodies have at least 80% sequence identity, more particularly atleast 90% sequence identity, even more particularly at least 95%sequence identity, most particularly at least 98% sequence identity,with the RHD5 antibody within their CDR regions. Additionally oralternatively, the antibodies have an amino acid sequence which differsfrom RHD5 in maximally 3 amino acids within each CDR. More particularly,the total number of modified amino acids within all of the CDRs is 15,even more particularly the maximum number of modified amino acids forall of the CDRs, compared to native RHD5, is 12.

Alternatively or additionally, a site on the C2 domain of Factor VIIImay also be partially inhibited. The present invention also includesligands other than polyclonal antibodies, in particular monoclonalantibodies, which reduce the release rate of Factor VIII from vonWillebrand factor. These monoclonal antibodies specifically targetFactor VIII when bound to von Willebrand factor and hence target anepitope associated with the complex of Factor VIII and von Willebrandfactor.

The antibodies of the present invention may be of human or animalorigin. They can be a result of a purposely directed immunization or canbe alloantibodies produced against exogenous Factor VIII.

The present invention further provides monoclonal antibodies havingsubstantially the same characteristics as the antibodies disclosedherein. Such antibodies can be produced by on purpose immunization inanimals, preferably in mouse, for instance by injecting human FactorVIII in mice and then fusing the spleen lymphocytes with a mouse myelomacell line, followed by identifying and cloning the cell culturesproducing anti-Factor VIII antibodies. The monoclonal antibodiesproduced in animals are then humanized, for instance by associating thebinding complementarity determining region (“CDR”) from the non-humanmonoclonal antibody with human framework regions—in particular theconstant C region of human gene—such as disclosed by Jones et al. inNature (1986) 321:522 or Riechmann in Nature (1988) 332:323. The presentinvention also relates to recombinant antibodies of antibodies accordingto the description hereof such as Krix-1 or RHD5, produced in anysuitable host cell (e.g. CHO cells).

The present invention also provides fragments of monoclonal antibodiessuch as Fab, Fab′, F(ab′)₂, scFv, CDR's, single variable domains as wellas derivatives, homologs and combinations of these. More particularly,these monoclonal antibodies and fragments may target a domain of FactorVIII, in particular the C1 domain of Factor VIII. They may alsopartially inhibit a site on the C2 domain of Factor VIII. They may alsotarget an epitope associated with the complex of von Willebrand factorand factor VIII. An aspect of the present invention is therefore toprovide ligands other than polyclonal antibodies which bind to a firstsite (e.g. in the C1 domain of Factor VIII) remote from a functionalsecond site (e.g. the site in the C2 domain of Factor VIII which isresponsible for binding phospholipids) in such a way that the functionof the second site is only partially impaired even when the ligand is ina molar and therapeutic excess.

Such fragments, which contain the antibody binding site, have lost anumber of properties of the parent antibody, such as complementactivation or capacity to bind to Fc gamma receptors. The presentinvention also includes single chain fragment variables (scFv), singlevariable domain fragments of the antibodies and combination of thesefragments and of the above mentioned fragments.

The present invention thus also provides fragments and derivatives, inparticular complementarity determining regions (“CDR's”) of themonoclonal anti-Factor VIII antibodies described herein as well ashomologs thereof For instance, the invention provides antigen-bindingfragments Fab, Fab′ and F(ab′)₂ generated by proteolytic digestion ofthe said monoclonal antibodies using methods well known in the art, suchas described by Stanworth et al., Handbook of Experimental Immunology(1978), vol. 1 chapter 8 (Blackwell Scientific Publications). Briefly,an F(ab′)₂ is obtainable after pepsin cleavage and is built up of bothlight chains and parts of the heavy chains disulfide linked via thehinge region. The Fab fragment is obtainable from the intact antibody orfrom the F(ab′)₂ by papain digestion of the hinge region and contains aone light chain and one part of the heavy chain. Fragments of antibodiescan also be obtained by synthesis or by recombinant methods described inthe art.

The invention also provides soluble or membrane anchored single-chainvariable parts (scFv fragments) of the above monoclonal antibodies.Methods for obtaining scFv fragments are known to the skilled person andinclude the method as follows. The DNA sequences of the variable partsof human heavy and light chains are amplified in separated reactions andcloned. A fifteen amino-acid linker sequence, for instance (Gly4 Ser)3,is inserted between VH and VL by a two-steps polymerase chain reaction(PCR), for instance according to Dieffenbach and Dveksler, “PCR Primer,a laboratory manual” (1995), Cold Spring Harbour Press, Plainview, N.Y.,USA. The resulting fragment is then inserted into a suitable vector forexpression of single chain fragment variable (scFv) as soluble orphage-displayed polypeptide. This can be achieved by methods well knownto those skilled in the art, such as described by Gilliland et al.,Tissue Antigens (1996) 47:1-20. The present invention also includes aligand comprising peptides representative of hypervariable regions of amonoclonal antibody which can be obtained by synthesis using an appliedbiosystem synthesizer, for instance a polypeptide synthesizer such asmodel 9050 available from Milligen (USA) or a model from a relatedtechnology, which alone or in combination with other or similarhypervariable regions will exert properties similar to that of theparent antibody.

The present invention further provides reshaped human monoclonalantibodies or human hybrid monoclonal antibodies against Factor VIII,which bind to and only partially inactivate Factor VIII or a complexincluding Factor VIII and von Willebrand factor which comprise onlyelements derived from the repertoire of human antibodies. Conventionallyin the art, until now it has only been possible to obtain antibodiesagainst Factor VIII derived from animals, e.g. mice, or to constructchimeric antibodies from human antibodies with the variable portionsderived from mice antibodies.

According to a further embodiment of the present invention, modifiedantibodies of the herein described monoclonal antibodies are provided,wherein the amino acid sequence is modified. Most particularly, modifiedversions of the partially inhibitory antibodies or fragments ofantibodies described herein are provided, whereby the modificationresults in a modified glycosylation pattern, whereby these modifiedantibodies are still capable of binding to Factor VIII and partiallyinactivating Factor VIII.

Methods for obtaining antibodies with a modified glycosylation are knownin the art and described herein. Briefly, such methods include thefollowing steps:

-   -   exposing antibodies to carbohydrate cleaving or transforming        enzymes;    -   producing the antibodies in cell lines with suitable        glycosylation enzymes or by modifying the cell culture        conditions to modify the activity of the glycosylation enzymes        of the cell line producing the antibodies    -   genetically modifying the antigen binding site of the antibody        in order to remove or introduce glycosylation sites, for example        by site-directed mutagenesis.        In a particular embodiment, said modified inhibitory antibodies        or fragments thereof have a modified glycosylation and, as a        result thereof, a modified (i.e. increased or decreased)        inhibitory activity. Most particularly, according to the present        invention the modified antibodies and fragments thereof have a        decreased inhibitory activity compared to the unmodified        antibody. The present invention thus discloses antibodies or        fragments thereof, derived from the antibodies of the present        invention, inhibiting Factor VIII activity by about 85, 50, 40,        30 and 20%. More particularly the invention relates to a        monoclonal antibody or fragment thereof, which is a modified        monoclonal antibody or fragment, inhibiting less than 65% of        Factor VIII activity.

According to a further particular embodiment, the modified inhibitoryantibodies of the present invention having a modified glycosylation arecharacterised in that the affinity of said antibodies or fragmentsthereof for their target protein is substantially unaffected compared tothe unmodified antibody. As indicated above, the invention also relatesto modified fragments and derivatives of the antibodies of the presentinvention. Thus, the modified antibodies of the present inventioninclude fragments thereof such as, but not limited to, Fab fragments,F(ab′)₂ fragments and scFvs.

The modification of the glycosylation of native antibodies can beobtained through different methods known in the art. Modification of theglycosylation pattern in the antigen binding site of the antibodies ofthe present invention can be achieved by enzymatic treatment of purifiedantibodies. Alternatively, modification of the glycans of the antibodiesof the present invention can be achieved by producing the antibodies incell lines with suitable glycosylation enzymes or by modifying the cellculture conditions to modify the activity of the glycosylation enzymesof the cell line producing the antibodies. Alternatively, the antibodiesof the present invention can also be produced by genetically modifyingthe antigen binding site of the antibody in order to remove or tointroduce glycosylation sites.

Many carbohydrate cleaving or transferring enzymes can be applied inorder to modify the glycosylation pattern of a native antibody. Theglycosylation can be increased or decreased completely or partially. Ina particular embodiment, the modification is obtained in the antigenbinding region of the antibody. Enzymes can be applied on a nativeantibody in a different order and under variable circumstances(concentrations, time, temperature, buffer, etc.) in order to obtainantibodies with different glycosylation patterns.

Enzymes such as peptide N-4(N-acetyl-beta-glucosaminyl)asparagineamidase F (PNGase F), also called N-glycosidase F can be used. Thisenzyme has a broad specificity, and it releases nearly all knownN-linked oligosaccharide chain from proteins (Plummer T H Jr et al.(1984) J Biol Chem. 259, 10700-10704). This enzyme releases tetra- andpenta-antennary chains. It is noteworthy that the activity of the enzymecan only be predicted when the glycoprotein is fully denatured.Accordingly, the activity of the enzyme on an intact antibody must becontrolled in each case. Methods to control the deglycosylation of theantibody are described in Current Protocols in Protein Science, Ed. G.Taylor, Unit 12.4; John Wiley & Sons, Inc.

In particular, the glycosylated and deglycosylated antibodies arecompared by isoelectrofocusing. Truncated glycoforms of IgG can begenerated by sequential enzymatic treatment as described in Mimura etal. (2001) J Biol Chem. 276, 45539-45547, and summarized in FIGS. 1 and2.

Sialic acids are the terminal sugars on many N- and O-linkedoligosaccharides. To remove sialic acid, the native IgG in acetatebuffer, pH 5.0, are exposed to sialidase (such as the sialidase fromArthrobacter ureafaciens, Roche Molecular Diagnostics, East Sussex, UK)at 37° C. for 24 h. Removal of sialic acids results in an increase inthe isoelectric point of the protein. IEF can therefore be used tocontrol removal of sialic acids.

Upon removal of sialic acids, galactose can be removed by treatementwith beta-galactosidase (Diplococcus pneumoniae, Roche MolecularBiologicals) in acetate buffer, at 37° C. for 24 h. Following removal ofsialic acid and galactose, N-acelylglucosamine can be cleaved bytreatment with N-acetyl-beta-D-glucosaminidase (D. pneumoniae, Roche,Molecular Biochemicals) in 37° C. for 24 h. Mannose residues can then beremoved by treatment with cc-mannosidase (jack bean, Glyko, Oxfordshire,UK) at 37° C. for 48 h (Mimura Y. et al. cited supra).

Different types of sialidase have also been described. The sialidase(neuraminidase) from Arthrobacter ureafaciens releases both alpha 2,3-and alpha 2,6-linked sialic acids, whereas the sialidase from theNewcastle disease virus releases only alpha 2,3 linked sialic acids(Jassal et al. (2001) Biochem Biophys Res Comm. 286: 243-249). Theendoglycosidase F2 cleaves the bound between the two GlcNAc residues inthe core region, leaving one GlcNAc still bound to the protein.Endoglycosidase F2 preferentially releases biantennary complex-typeoligosaccharides chains from glycoproteins but does not cleave tri- ortetraantennary chains

Endoglycosidase F3 is another endoglycosidase with a narrow substraterange: it cleaves triantennary chains. A core fucosylated biantennarychain is the only other demonstrated substrate. It does not cleavehigh-mannose hybrid, nonfucosylated biantennary or tetraantennarychains. All linkages which can be cleaved by endoglycosydase F2 and F3are not exposed in a mature antibody. Methods suitable to determinewhether an antibody can be usefully modified by these endoglycosidaseinclude SDS-PAGE, lectin binding methods using Ricinus communisagglutinin-1 and IEF as described above. Conversely, glycan residues canbe enzymatically added to carbohydrate expressed in the variable part ofthe antibody. For example, treatment with sialidase as described abovecan be followed by treatment with galactosyl-transferase and UDP-Gal ina suitable buffer (Krapp et al. (2003) J Mol Biol. 325, 979-989). Themodified antibodies are then homogenous for galactosylation of thecarbohydrate chain (biantennary digalactosylated glycoform).

The purification of antibodies carrying different oligosaccharides isalso known to persons skilled in the art. The antibodies carryingdifferent oligosaccharides can be purified by lectin affinitychromatography, such as Concanavalin A (binding to a bisecting GlcNAc).Aleuria aurantia differentiates on the basis of core fucosylation.Ricinus communis agglutinin 1 fractionates according to the number ofgalactose residues because this lectin exhibits specific affinity tooligosaccharides ending with galactose (Youings et al. (1996) Biochem J.314, 621).

An alternative method for modifying the glycosylation of an antibody isto generate recombinant antibodies with modified glycosylation patternby producing recombinant antibodies in cell lines selected as a functionof their repertoire of glycosylation enzymes. Chinese Hamster Ovarycells (CHO) are well known example of such a cell line.

Although CHO cells have most of the human repertoire of glycosylationenzymes, they are deficient in particular glycosyltransferases. Inparticular, the alpha 2,6-sialyl-transferase gene (1,2) is not expressedendogenously in CHO cells. This enzyme adds terminal galactose sugarswith sialic acid in the alpha 2,6 position on the Gal beta 1, 4G1cNAc-Rsequence. However, CHO cells express a functional alpha2,3-sialyl-transferase so that the terminal sialic acids are in alpha2,3 linkage to galactose. Alpha-3/4 fucosyltransferase is also notsynthesized by these cells (Grabenhorst et al. (1999) Glycoconj. J. 16,81).

Another method to produce recombinant antibody with modifiedglycosylation pattern is to use a cell line genetically modified toexpress glycosylation enzyme from other strains. In particular, a CHO-K1cell line transfected with an alpha 2,6-sialyltransferase gene clonedfrom another strain can be used (cited supra).

Any expression system is potentially suitable for the generation ofrecombinant antibody with modified glycosylation pattern such as yeast(for example Saccharomyces, Pichia, Hansenula), insect cells(baculovirus expression), plant cells or plants, or mammalian cells. Forthe expression of fragments of an antibody yeast expression provide analternative for insect or mammalian cell expression. If no glycosylationat all is needed, the expression in bacteria is considered.

With respect to yeasts, the methylotrophic yeast Pichia pastoris wasreported to attach an average of 8 to 14 mannose units, i.e.Man(8-14)GlcNAc(2) per glycosylation site (Tschopp in EP0256421) andapproximately 85% of the N-linked oligosaccharides are in the size rangeMan(8-14)GlcNAc(2) (Grinna and Tschopp (1989) Yeast 5,107-115.).Aspergillus niger is adding Man(5-10)GlcNAc(2) to N-glycosylation sites(Panchal and Wodzinski (1998) Prep Biochem Biotechnol. 28, 201-217). TheSaccharomyces cerevisiae glycosylation deficient mutant mnn9 differsfrom wild-type S. cerevisiae in that mnn9 cells produce glycosylatedproteins with a modified oligosaccharide consisting ofMan(9-13)GlcNAc(2) instead of hyperglycosylated proteins (Mackay et al.in U.S. Pat. No. 5,135,854), However, characteristic for S. cerevisiae(wild-type and mnn9 mutant) core oligosaccharides is the presence ofterminal alpha1,3-linked mannose residues (Montesino et al. (1998)Protein Expr Purif. 14, 197-207.). Oligosaccharides attached toN-glycosylation sites of proteins expressed in P. pastoris or S.cerevisiae och1mnn1 are devoid of such terminal alpha1,3-linked mannoses(Gellissen et al. (2000) Appl Microbiol Biotechnol. 54, 741-750).Terminal alpha1,3-linked mannoses are considered to be allergenic(Jenkins et al. (1996) Nat. Biotechnol. 14, 975-981). Therefor, proteinscarrying on their oligosaccharides terminal alpha1,3-linked mannoseresidues are likely not suitable for diagnostic or therapeutic purposes.

The repertoire of glycosylating enzymes differs from cell type to celltype. In order to obtain a desired glycosylation pattern one or moreglycosylating enzymes can be (over)expressed by transient or stabletransfection. Equally one or more glycosylating enzymes can betemporarily (for example by antisense or siRNA technology) orpermanently inhibited (gene inactivation). In certain embodiment yeastcells are used which have a limited repertoire of enzymes involved inglycosylation. Herein one or more human genes involved in glycosylationcan be introduced to obtain a desired glycosylation pattern.

Additionally or alternatively, culture conditions can be exploited tomodify the glycosylation of the recombinant antibody. The concentrationof dissolved oxygen at steady state in serum free culture has an effecton glycosylation of antibody. The extent of galactosylation is reducedwith reduced dissolved oxygen concentrations (Kunkel et al. (1998). JBiotechnol. 62, 55-71). Supplementing the medium with more than 20 mMN-acetylglycosamine can also induce new antibody glycoforms (Tachibanaet al. (1992). Biochem Biophys Res Commun. 189, 625-32; Tachibana et al.(1996) In Vitro Cell Dev Biol Anim. 32, 178-183). Glucocorticoidhormones and interleukin 6 are involved in the modulation of proteinglycosylation (Canella and Margni (2002) Hybrid Hybridomics 21, 203).Other factors which influence glycosylation are changes in the pH ofculture medium and the availability of precursors and nutrients.

Another alternative to the enzymatical modifications and the recombinantproduction of the antibodies is to use (site-directed) mutagenesis. Newglycosylation sites can be introduced or existing glycosylation sitescan be removed with this technique. N-glycosylation sites can beintroduced by site directed mutagenesis in the variable region of theantibody. Preferably, the mutations are introduced as single amino acidchanges, to minimize the effect of the amino acid substitution on theaffinity of the antibody for the antigen. Addition of andN-glycosylation site is performed by creation of an Asn-X-Ser/Thrsequence, most commonly by mutating a codon to encode Asn. Moreover, itis preferable that the sites for additional glycosylation are selectedat positions predicted to be accessible to glycosyltransferases.Alternatively, amino-acid stretches containing N-glycosylation sites canbe selected in the published sequences of antibodies glycosylated in thevariable region. The selection of antibodies inhibiting Factor VIIIactivity in a desirable manner can be made using the Bethesda assay(Kasper et al. (1975) cited supra). The protein structure can also bemodified to indirectly modify glycosylation (Lund et al. (1996) JImmunol. 157, 4963, Lund et al. (2000), Eur J Biochem. 267, 7246).Site-directed mutagenesis is a method well-known to the person skilledin the art, and include the Zoller and Smith method (Zoller and Smith(1987) Methods Enzymol. 154:329-50).

In the context of the present invention, the modifications in theglycosylation of the antibodies most particularly occur in the variableregion (i.e. VH and/or VL) of the antibodies. In a more particularembodiment, the modified antibody is a modified Krix-1 antibody, with amodified N-glycosylation in the variable region, more particularly witha mutated glycosylation site at positions Asn47 to Thr49 of the heavychain, more in particular with heavy chain Asn47 changed to G1n47(KRIX-1Q), G1u47 (KRIX-1E) or Asp47 (KRIX-1D) and/or heavy chain Thr49to A1a49 (KRIX-1A).

According to a further aspect of the invention, sets of at least twoantibodies and/or antibody fragments, capable of binding the sameantigen and demonstrating variable maximal inhibition of Factor VIII aredisclosed. According to a particular embodiment, the set of at least twoantibodies comprises the unmodified antibody or a fragment thereof andat least one modified version thereof, or a fragment thereof.Alternatively, the set of at least two antibodies comprises two modifiedversions of the anti-Factor VIII antibody of the invention.

According to a further aspect, the present invention provides cell linesproducing human monoclonal antibodies which are reactive with humanFactor VIII and more specifically have the capacity of inactivating theco-factor activity of human Factor VIII by interfering with proteolyticcleavage site or von Willebrand factor or tenase complex reaction or byinducing a three-dimensional conformational change in Factor VIII, inparticular by targeting a domain of Factor VIII and by recognizingepitopes located in the said domain.

According to a particular embodiment, the present invention providescell lines producing antibodies directed against the C1 domain of FactorVIII.

The cell line named KRIX-1 producing monoclonal antibodies according tothe present invention was deposited with the BCCM /LMBP (BelgianCo-ordinated Collections of Microorganisms/Plasmid CollectionLaboratorium voor Moleculaire Biologie, University of Ghent K. L.Ledeganckstraat 35, B-9000 Ghent, BE under accession number LMBP 5089CBon Jul. 1, 1999. The cell line named RHDS also producing monoclonalantibodies according to the present invention was deposited with theBelgian Coordinated Collections of micro-organisms, under accessionnumber LMBP 6165CB, by the D. Collen Research Foundation on *** August2004.

The present invention further provides cell lines producing humanmonoclonal antibodies having a reactivity substantially similar to thatof the human monoclonal antibodies obtained from the above-mentioneddeposited cell lines, as well as the human monoclonal antibodiesobtainable from these further cell lines, and fragments and derivativesthereof. The present invention thus provides more in particular celllines producing monoclonal antibodies which bind to the C1 domain ofFactor VIII; more particularly the antibodies of the invention bind tothe same antigen, more in particular to the same epitope, bound by theantibody produced by cell lines mentioned hereinabove; even moreparticularly, the present invention provides antibodies which competewith the antibodies disclosed herein for binding to Factor VIII astested with an ELISA, as described herein.

According to the present invention ligands, other than polyclonalantibodies are provided, having the capacity of only partiallyinactivating a factor (or a complex including a factor) involved inhemostasis, in particular in the coagulation cascade of blood, mostparticularly Factor VIII or a complex including Factor VIII. Suchpartial inactivation is ensured by these ligands by binding to a site ofthe said factor or complex, whereby the said partial inactivation alsooccurs place when the ligand of the invention is in a molar excess withrespect to the said factor. The site to which the ligand binds may ormay not be directly or substantially involved in a physiologicalinteraction of the said factor or complex. For instance, the ligand maybind to a site which is at a predetermined distance away from aphysiologically functional site of the said factor.

According to a particular embodiment, the “partial” inhibition or“plateau” inactivation of the ligands or antibodies of the inventioncorresponds to at most 98% inactivation, preferably an at most 95%inactivation, as determined by a suitable test method such as forinstance the chromogenic assay available from Coatest® (Kabi Vitrum,Brussels, Belgium) or from Chromogenix AB, Mölndal (Sweden).Alternatively, inhibition of Factor VIII activity can be measured bydetermining blood clotting in the Bethesda assay (Kasper et al. (1975).The level of activation required may depend upon the physiologicalfunction of the factor involved in hemostasis.

According to a particular embodiment of the invention, the partialinhibition achieved by the antibodies of the present invention is atleast 10%, most particularly at least 20%. According to a particularembodiment of the invention, inactivation of the blood factor is atleast about 65%, alternatively at least about 70%, as determined by thesame test method as above. It will be appreciated that the ligands inaccordance with the present invention operate in a different way fromthe mechanism conventionally ascribed to type II antibodies againstFactor VIII. One conventional mechanism is that of competition withanother factor, e.g. von Willebrand factor. The kinetics of acompetition mechanism mean that if the one species is at a highconcentration compared with the other (e.g. in a molar excess), theinhibition is effectively complete. In contrast, the ligands of thepresent invention reach a plateau in the inactivation of the relevantfactor, which is substantially independent of the excess of the ligand.The other conventional mechanism ascribed to type II antibodies is thatof low affinity: also in this case, an excess will drive the reaction tocomplete inhibition.

According to yet another aspect of the invention, two or more antibodiesor antibody fragments with different partial inhibitory activity ofFactor VIII can be combined resulting in a mixture with an intermediateinhibitory activity for Factor VIII. A particular embodiment of theinvention is a mixture of two or more partially inhibitory antibodiesagainst Factor VIII or fragments thereof, which ensures a given partialinhibition of Factor VIII, whatever the excess of the mixture ofantibodies over Factor VIII. Using combinations of different inhibitoryantibodies and/or fragments thereof in specific ratios, mixtures withspecific inhibitory activity of Factor VIII can be obtained. Thus, thepresent invention relates to a combination of two or more antibodies orantibody fragments with different partial inhibitory activities.According to a specific embodiment, two different antibodies orfragments thereof with different inhibitory activity are combined.According to a further specific embodiment the native antibody iscombined with one or more antibodies derived therefrom or fragmentsthereof having a lower inhibitory activity. According to a furtherembodiment of this aspect of the invention, the native antibody iscombined with an antibody or antibody fragment having a modifiedglycosylation with respect to the native antibody, derived therefrom.Such combinations or mixtures are of interest for further adjustment ofthe inhibitory activity of the antibody, e.g. in the development ofpatient-specific pharmaceuticals. The present invention further showsthat mixtures of inhibitory antibodies, being derived from the samenative unmodified antibody, with different individual inhibitoryactivity, result in a mixture wherein an intermediate inhibitoryactivity is obtained. According to a particular embodiment such as amixture comprises the unmodified monoclonal Krix-1 antibody or afragment thereof, and a modified Krix-1 antibody, or a fragment thereof.A further embodiment provides a mixture comprising two differentmodified versions of the Krix-1 antibody. Alternatively, the inventionprovides mixtures of different antibodies which recognize the sameantigen, and thus are competitive for each other. According to aparticular embodiment such as a mixture comprises the monoclonal Krix-1antibody (first antibody)or antibody fragment or a modified versionthereof and a second monoclonal antibody or antibody fragment obtainedfrom the RHD5 cell line or a modified version thereof

The invention further provides a pharmaceutical composition for theprevention or treatment of disorders of hemostasis, in particular of thecoagulation cascade and resulting thrombotic pathologic conditions inhumans, comprising, as an active ingredient, a ligand other than apolyclonal antibody, preferably a human monoclonal antibody such asdisclosed hereinabove, in admixture with a pharmaceutically acceptablecarrier.

According to a particular embodiment the pharmaceutical compositioncomprises a monoclonal antibody which is a human monoclonal antibody, ora fragment, derivative or homolog thereof such as a modified versionthereof, obtainable from the cell line KRIX-1 deposited with the BelgianCo-ordinated Collections of Micro-organisms under accession number LMBP5089CB or from the cell line RHD5 deposited with the BelgianCo-ordinated Collections of Micro-organisms under accession number LMBP6165CB. According to a particular embodiment, the pharmaceuticalcompositions of the present invention comprises a monoclonal antibody,or a fragment thereof which is a modified version of the antibodiesdescribed herein, more particularly a modified version having at least80% sequence identity with antibodies described herein.

The degree of sequence identity or similarity with the said monoclonalantibody is preferably at least 80%, more preferably 90% even morepreferably 95%, and most preferably at least 99% and the sequenceidentity or similarity is preferably particularly in respect to thevariable regions (variable heavy and/or variable light regions), mostparticularly in respect to the complementarity determining regions(CDRs) of the antibody. Additionally or alternatively the degree ofidentity with the monoclonal antibody is expressed as having maximally 3different amino acids within each CDR compared to Krix-1. In aparticular embodiment, the total number of amino acids that can bechanged within all the CDRs is 12. It will be understood that, withinthe framework regions in the variable regions and within the constantregions, the sequence identity or similarity with Krix-1 is lesscritical, as these regions affect binding to the antigen to a lesserextent.

A ligand or antibody in accordance with the present invention may alsoinclude a synthetic polypeptide of equivalent potency. Thepharmaceutical composition of the present invention should comprise atherapeutically effective amount of the said above ingredient, such asindicated hereinafter in respect to the method of treatment orprevention.

The pharmaceutical composition of the present invention may furthercomprise, namely in view of a so-called adjunctive anti-thrombotictreatment, a therapeutically effective amount of a thrombolytic agent.Such thrombolytic agents, as well as their usual dosage depending on theclass to which they belong, are well known to those skilled in the art.Among numerous examples of thrombolytic agents which may be included inthe pharmaceutical compositions of the invention, the followingnon-limiting list may be particularly cited: t-PA, streptokinase,reptilase, TNK-t-PA or staphylokinase.

The pharmaceutical composition of the present invention may furthercomprise a pharmaceutical carrier. Suitable pharmaceutical carriers foruse in the pharmaceutical compositions of the invention are describedfor instance in Remington's Pharmaceutical Sciences 16^(th) ed. (1980)and their formulation is well known to those skilled in the art. Theyinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents (for example phenol, sorbic acid, chlorobutanol),isotonic agents (such as sugars or sodium chloride) and the like.Additional ingredients may be included in order to control the durationof action of the monoclonal antibody active ingredient in thecomposition. Control release compositions may thus be achieved byselecting appropriate polymer carriers such as for example polyesters,polyamino acids, polyvinyl pyrrolidone, ethylene-vinyl acetatecopolymers, methylcellulose, carboxy-methylcellulose, protamine sulfateand the like. The rate of drug release and duration of action may alsobe controlled by incorporating the monoclonal antibody active ingredientinto particles, e.g. microcapsules, of a polymeric substance such ashydrogels, polylactic acid, hydroxymethylcellulose, polymethylmethacrylate and the other above-described polymers. Such methodsinclude colloid drug delivery systems like liposomes, microspheres,microemulsions, nanoparticles, nanocapsules and so on. Depending on theroute of administration, the pharmaceutical composition comprising theactive ingredient may require protective coatings. The pharmaceuticalform suitable for injectionable use include sterile aqueous solutions ordispersions and sterile powders for the extemporaneous preparationthereof. Typical carriers therefore include biocompatible aqueousbuffers, ethanol, glycerol, propylene glycol, polyethylene glycol andmixtures thereof.

The present invention also provides the use of a ligand, other than apolyclonal antibody (as disclosed above) as a medicament. Morepreferably the medicament used in the present invention is a means forpreventing and/or treating disorders of hemostasis, in particular,coagulation disorders and other thrombotic pathologic conditions inmammals, preferably in humans. The said ligand may be provided to apatient by any means well known in the art, i.e. orally, intranasally,subcutaneously, intramuscularly, intradermally, intravenously,intraarterially, parenterally or by catheterization. According to thepresent invention, the ligand may also be used as a medicament inconjunction or association with another medicament, for instance athrombolytic agent such as disclosed hereinabove under the heading ofpharmaceutical compositions.

The present invention therefore provides a method of treatment and/orprevention of hemostasis, coagulation disorder or thrombotic pathologiccondition as well as a method of attenuation of coagulation in a mammal,preferably a human, comprising administering to a mammal in need of suchtreatment or prevention or attenuation of coagulation a therapeuticallyeffective amount of a ligand or antibody other than a polyclonalantibody such as disclosed hereinabove.

According to a particular embodiment the method of treatment and/orprevention comprises the administration of a human or humanizedmonoclonal antibody obtainable from cell line KRIX-1 deposited with theBelgian Co-ordinated Collections of Micro-organisms under accessionnumber LMBP 5089CB or an antigen-binding fragment Fab, Fab′ or F(ab′)₂,a complementarity determining region (CDR), a soluble ormembrane-anchored single-chain variable part (scFv), a single variabledomain or a derivative, such as a modified antibody, more particularly aglycosylation-modified antibody or combination of any of these elements.Alternatively, the method of treatment and/or prevention comprises theadministration of a human or humanized monoclonal antibody obtainablefrom cell line RHD5 deposited with the Belgian Coordinated Collectionsof micro-organisms, under accession number LMBP 6165CB, or anantigen-binding fragment Fab, Fab′ or F(ab′)₂, a complementaritydetermining region (CDR), a soluble or membrane-anchored single-chainvariable part (scFv), a single variable domain or a derivative, such asa modified antibody, more particularly a glycosylation-modified antibodyor combination of any of these elements. In yet a further particularembodiment, the method of treatment and/or prevention comprises theadministration of ligand which is a human or humanized monoclonalantibody binding to the antigen, more in particular to the epitope,bound by the antibody produced by Krix-1 cell line or the RHD5 cellline. In another particular embodiment, the antibody or fragment thereofused in the methods of the present invention is a human or humanizedmonoclonal antibody which competes with Krix-1 or with RHD5 for bindingto Factor VIII as tested with an ELISA as described herein. In anotherparticular embodiment, the antibody or fragment thereof used in themethods of the present invention is a human or humanized monoclonalantibody binding to the C1 domain of Factor VIII, as well as fragments,derivatives, and homologs thereof, with the amino acid sequence of itsvariable heavy chain being at least 80% (more in particular 90% or 95%)identical or similar to the amino acid sequence of the variable heavychain of the described anti-Factor VIII antibodies of the invention,within the CDRs of said variable heavy chain and/or a variable lightchain sequence being at least 80% (more in particular 90% or 95%)identical to the amino acid sequence of the variable light chainsequence of known anti-Factor VIII antibodies of the invention, withinthe CDRs of said variable light chain. Additionally or alternatively,the antibodies used in the methods of the invention are characterized inthat, with regard to the CDR regions, they differ from the knownanti-Factor VIII antibodies in maximally three amino acids within eachCDR. Most particularly, the total number of amino acid differenceswithin the CDRs is 12. In another particular embodiment, the antibody orfragment thereof used in the methods of the present invention is amonoclonal antibody, in particular human or humanized, as well asfragments, derivatives, and homologs of the antibodies described in thepresent invention and comprises

-   -   a variable heavy chain sequence comprising CDRs being at least        80%, more in particular 90% or 95%, identical to the amino acid        sequence of the CDRs of the variable heavy chain sequence of        Factor VIIIKrix-1; and/or    -   a variable light chain sequence comprising CDRs being at least        80%, more in particular 90% or 95%, identical to the amino acid        sequence of the CDRs of the variable light chain sequence of        Factor VIIIKrix-1.        According to a particular embodiment, d ligands used in the        methods of the present invention are fragments and derivatives,        including modified antibodies such as deglycosylated antibodies,        in particular complementarity determining regions (“CDR's”) of        the herein described monoclonal anti-Factor VIII antibodies as        well as homologs thereof.

A therapeutically effective amount as used herein means from about 1microgram to about 10 milligrams per kilogram of body weight, morepreferably from about 10 micrograms to about 1 milligram per kilogram ofbody weight of the mammal to be treated. It will be appreciated that, inview of the long half-life time of most IgG human antibodies, theligands of the present invention which are monoclonal antibodies of thesaid class will enjoy a periodicity of treatment which participates inthe comfort of the patient.

The present invention provides methods of treatment and prevention aswell as pharmaceutical compositions for the treatment or prevention of athrombotic pathological condition. Particular embodiments of the saidthrombotic pathologic condition to be prevented or treated, include butare not limited to intravascular coagulation, arterial thrombosis (whichmay be responsible for acute myocardial infarction and stroke),peripheral artery disease, coronary arterial disease, arterialrestenosis, venous thrombosis (which commonly occurs in peripheral veinsas a consequence of accidental or surgical trauma or immobilization) orarteriosclerosis. When referring to the treatment or prevention ofthrombotic pathological conditions, the disorders outlined herein aboveare thus envisaged

A number of conditions are known to increase of the risk of thrombusformation such as arterial fibrillation, vascular interventions,mechanical heart valves, heart attack, unstable angina, acute ischemicstroke. Thus, it can be envisaged that the ligands or antibodiesdescribed herein are particularly suited for preventing a thromboticpathological condition, when these conditions occur.

In a particular embodiment of the method of treatment of the presentinvention, the patient is provided with a bolus (intravenously injected)at a dosage determined by the ordinary skilled physician depending oncriteria which establish the particular patient's clinical condition.

The method of treatment and/or prevention according to the invention mayinclude further treatment or prevention of the same thromboticpathologic condition by administrating, preferably by sequentiallyadministrating, to the patient a therapeutically effective amount of athrombolytic agent such as disclosed hereinabove under the heading ofpharmaceutical compositions. Sequentially, as used herein, means thatthe ligand of the present invention and the known thrombolytic agent areadministered to the patient sequentially but not simultaneously.

According to yet a further aspect, the present invention providesmethods for the identification and/or selection of antibodies which arecapable of partially inhibiting a wild type protein, more particularlyFactor VIII, with the plateau effect described herein.

The conventional technique of immunizing an animal such as a mouse witha protein such as Factor VIII elicits an immunological response whichmay involve several epitopes on the Factor VIII molecule. Even whenusing specific antigens which are related to the activity of FactorVIII, the likeliness of obtaining an antibody which partially inhibitsFactor VIII is limited. The present invention provides more selectivemethods of obtaining monoclonal antibodies capable of only partiallyinhibiting a wild-type protein. First, a donor, e.g. a mammal such as ahuman, is provided (i.e. selected) which has a partially functional,modified version of a wild-type protein. The said modification, whichlies in a domain of the protein, may be due to any cause, e.g. race orvariety, to genetic defects at birth, to an illness or by humaninterference, e.g. immunotolerance against the functionally modifiedversion. The mammal donor is then administered the wild-type protein inorder to elicit an immune response; at this stage, it is important thata sufficient quantity of the wild-type protein (e.g. Factor VIII) beadministered until an immune response is generated in the mammal donor.Then, in a final step of the method, selection of B-cells from themammal donor will result in a much greater chance of obtainingmonoclonal antibodies against an epitope in the region of themodification, as this region of modification in the wild-type protein isrecognized as foreign by the mammal host. As this region of modificationis responsible for the partial inhibition of function of the protein inthe mammal donor, it is likely that antibodies directed to this regionin the wild-type protein will also affect the function of the protein.When applied to Factor VIII, this method will result in a greater chanceof obtaining partially inhibitory antibodies to Factor VIII. Such amethod of obtaining monoclonal antibodies from a non-human mammalagainst a domain of a protein thus comprises the steps of:

-   -   a) selecting a non-human mammal having a modified and at least        partially functional (or partly physiologically active) protein,        the modification being with respect to a wild type protein and        lying in the relevant domain of the protein;    -   b) administering the wild type protein to the non-human mammal        in order to elicit an immune response, and    -   c) selecting B-lymphocytes from the non-human mammal which        produce antibodies which only partially inactivate the wild type        protein.        According to this method, the standard practice is to sacrifice        the non-human mammal and to remove its spleen in order to        perform step (c).

The present invention additionally provides a method of obtainingmonoclonal antibodies from the blood of a human being having a modifiedand (at least) partially functional physiologically active protein, themodification being with respect to a wild type protein and lying in adomain of the protein, and to whom the wild type protein wasadministered, the said method comprising the step of selecting, from theblood of said human being, B-lymphocytes which produce antibodies whichonly partially inactivate the wild type protein.

As described above, the present invention provides monoclonal antibodiesdirected to the C1 domain of Factor VIII and capable of partiallyinhibiting Factor VIII, whereby a number of specific antibodies areprovided. Additionally, antibodies capable of competing with thesepartially inhibiting Factor VIII antibodies are also envisaged. Methodsfor identifying such antibodies capable of competing with the antibodiesof the invention are known in the art. According to one embodiment ofthe invention, competing partially inhibitory Factor VIII antibodies areidentified by a method which comprises the steps of contacting FactorVIII or a fragment of Factor VIII comprising the C1 domain with a firstinhibitory antibody and a candidate inhibitory antibody, and assayingthe capacity of said candidate antibody to compete with the binding ofthe Factor VIII inhibitory antibody said Factor VIII or fragment ofFactor VIII. Said candidate antibody can be obtained by the methodsdescribed herein above or can be an uncharacterised antibody, which ispart of a large pool of antibodies, each of which are screened by thecompetition assay. Alternatively, the uncharacterised antibody is firstscreened for its ability to bind factor VIII. This can be done forexample by incubating the uncharacterized antibodies with factor VIIIfixed on microtiter plates, whereafter labelled known Factor VIIIinhibitory antibody, such as Krix-1, is added and assayed for itsbinding to Factor VIII. Alternatively, the known Factor VIII inhibitoryantibody and the candidate antibody are mixed together before assayingthe residual binding of the known inhibitory antibody to Factor VIII. Ina particular embodiment of the invention the known inhibitory FactorVIII antibody used for the identification of novel, competing partiallyinhibitory antibodies is Krix-1 or RHD5, or fragments or derivativesthereof. According to a further particular embodiment of the inventionthe antibodies obtained in such a way are further screened for theirFactor VIII inhibitory activity and for their plateau-effect.

The present invention thus also relates to (purified) antibodiesobtained from this method which can be used in mixtures together withKrix-1, RHD5 or modified versions thereof according to the presentinvention and in the generation of pharmaceutical compositions.

The present invention, as embodied in the various above disclosedaspects, has a number of advantages. The major advantage of thetherapeutic use of the human monoclonal antibodies of the invention isthat the treatment is highly specific for the immune response underconsideration. In hypercoagulation states, the specificity of the humanmonoclonal antibodies of the invention ensures that interaction withinthe coagulation cascade pathway is limited to the factor recognized bythe antibody.

More specifically, the use of the anti-Factor VIII antibodies having theabove-mentioned preferred characteristics brings a unique combination ofthe advantages related to the targeting of Factor VIII, those related tothe characteristics of Factor VIII inhibition and those related to theuse of antibodies:

targeting Factor VIII means that neutralizing a co-factor activity suchas that of Factor VIII carries no risk of completely inhibiting theenzymatic activity it enhances, thereby representing an advantage overmethods targeting directly enzymes such as Factor IX.

the embodiments of the inhibitors described above have in common thatthey efficiently but only partially inhibit the co-factor activity ofFactor VIII, setting a therapeutically useful plateau, even when themonoclonal antibody of the invention is used in more than 100-foldexcess. Monoclonal antibodies in accordance with the present inventionachieve a plateau effect in inactivation of Factor VIII, allowing bolusapplication, yielding safe antithrombotic protection over several weekswithout the need of monitoring or the risk of overdosage.

where human IgG is used, an additional advantage is obtained. Human IgGantibodies exhibit a prolonged half-life time of three weeks (except forIgG3 which is one week), thus providing very stable plasma levels of theanti-thrombotic agent and allowing for a drastic reduction in thefrequency of administration.

Further, the use of human antibodies or derivatives carries a minimalrisk of inducing immune responses.

The present invention relates to ligands or antibodies capable ofpartially inhibiting the activity of Factor VIII. As indicated herein,these are useful in the treatment of diseases in which the formation ofblood clots is pathological. The effects of the reagents of the presentinvention on blood clotting is commonly evaluated using ‘activatedProthrombin time’ (aPTT).

The activated partial thromboplastin time (PTT) measures the clottingtime from the activation of Factor XII, through the formation of fibrinclot. This measures the integrity of the intrinsic and common pathwaysof coagulation, whereas the prothrombin time (PT) measures the integrityof the extrinsic and common pathways of coagulation. PTT prolongationsare caused by either factor deficiencies (especially of Factors VIII,IX, XI, and/or XII), or inhibitors (most commonly, lupus anticoagulants,or therapeutic anticoagulants such as heparin, hirudin, or argatroban).PTT results are reported as the number of seconds the blood takes toclot when mixed with a thromboplastin reagent. The normal values of aPTTrange between around 25 to 35 seconds. In patients receivinganticoagulant therapy the aPTT value will be 1.5 to 2.5 times thecontrol values. The International Normalized Ratio (INR) was created bythe World Health Organization because PTT results can vary depending onthe thromboplastin reagent used. The INR is a conversion unit that takesinto account the different sensitivities of thromboplastins. The INR iswidely accepted as the standard unit for reporting PT results.

The effectiveness of anticoagulants, more particularly coagulants takenorally, can vary over time, as changes in diet, (particularly foods highin vitamin K), alcohol use, other drugs and illness can all affect PTT.These factors require that the PTT is monitored regularly so the patientstays within the desired therapeutic range. Oral anticoagulant dosagesare then adjusted according to the results of the PT test.

The present invention is further described by the following exampleswhich are provided for illustrative purposes only. The Krix-1 antibodyreferred to herein above is also referred to as the KRIX-1 antibodyhereunder.

Example 1 Production of Monoclonal Antibodies Derived from Hemophilia APatients

Human monoclonal antibodies of the desired specificity andcharacteristics are produced by transformation of B lymphocytes obtainedfrom the peripheral blood of patients suffering from hemophilia A oracquired hemophilia. The method of selecting patients is an embodimentof the present invention. In order to elicit a more specificimmunological response, patients are sought who have an impairedfunction of a physiologically active protein, e.g. Factor VIII. By“impaired” is meant that some residual function is available but thatthis is less than is known for the wild-type of the same protein. Acomparison between the self-protein and the wild-type protein shouldexhibit a difference in the two proteins, preferably in a region ordomain which is of interest. The difference may be a deletion or asubstitution of one or more amino acids with others. The patients arethen administered enough of the wild-type protein to elicit animmunological response. Then, B-lymphocytes are extracted from thepatients and selected based on the production of antibodies which havedesirable properties. Although reference is made to “patients” above,the method in accordance with this embodiment may be applied generallyto mammals. The above procedure results in a greater chance of obtainingantibodies which target the domain containing the defect.

B cells are transformed by infection with the Epstein-Barr virus andactivation of surface antigens using techniques well known by thoseskilled in the art. Cell supernatants containing appropriate antibodiesare identified by a specific assay procedure such as described in moredetails hereinbelow.

Thus, antibodies towards Factor VIII are identified by reacting thesupernatant with polystyrene microtitration plates coated with FactorVIII or with Factor VIII/von Willebrand factor complexes. The binding ofspecific antibodies is detected by addition of a non human IgG reagentcoupled to an enzyme. Addition of an enzyme substrate which is convertedto a colored compound in the presence of the enzyme allows the detectionof specific antibodies. Such methods referred to as enzyme-linkedimmunoassays (ELISA) are well known to those skilled in the art anddescribed in details e.g. in Current Protocols in Immunology, chapter 2,John Wiley & Sons (1994), the content of which is incorporated herein byreference.

More specifically in the present case, the binding of anti-Factor VIIIIgG antibodies was detected by addition of a horseradish peroxidaselabeled mouse monoclonal antibody specific for human Fc gamma The IgGsubclass of the anti-Factor VIII antibody was detected in ELISA, aspresented in FIG. 1. The inhibition of Factor VIII functional activitywas tested in a functional coagulation assay as follows. Equal volume ofcell culture supernatant and of a pool of normal plasma were incubatedfor two hours at 37° C. and the residual Factor VIII activity measuredthereafter. Those antibodies which significantly inhibit Factor VIIIactivity are shown with an asterisk in FIG. 1.

B cells (such as BO 2C11) producing anti-Factor VIII antibodies are thenexpanded and cloned by limiting dilution as described for instance inCurrent Protocols in Immunology (see supra). Anti-Factor VIII antibodieshaving the capacity to inhibit the procoagulant activity of Factor VIIIas described above are identified using a chromogenic assay kit such asa Factor VIII chromogenic assay from Dade, Düchingen, Germany orCoatest® commercially available from Kabi Vitrum (Brussels, Belgium) orChromogenix AB (Mölndal, Sweden).

Equal volumes of monoclonal antibodies BO 2C11 and a pool of normalblood plasma were incubated for 2 hours at 37° C. BO 2C11 concentrationsbefore mixing are shown on the X axis. The reduction of Factor VIIIactivity was measured in a coagulation assay and was expressed as apercentage of the activity obtained in the absence of antibody (see FIG.2). The residual activity goes to zero asymptotically (completeinhibition).

Antibodies which inhibit Factor VIII function with sufficient affinitybut do not inhibit Factor VIII pro-coagulant activity completely, evenwhen used in large antibody excess, were selected in a furtherembodiment of the present invention. A representative example of such anantibody is provided in FIG. 3 where, equal volumes of KRIX-1 and ofrecombinant Factor VIII or of normal plasma being incubated for twohours at 37° C. and concentrations (expressed in microgr/ml) of KRIX-1before mixing with plasma being as indicated, the residual Factor VIIIactivity was measured using the above-mentioned chromogenic assay. FIG.3 interestingly shows about 60% Factor VIII inhibition at aconcentration of 0.1 microgr/ml and more interestingly an asymptoticFactor VIII inhibition of about 80% in the whole range of concentrationsfrom 0.5 to 300 microgr/ml.

The thus selected antibodies are then produced in bulk culture andpurified by affinity chromatography using methods well known to thoseskilled in the art.

The details of a non-limiting preparation technique are as follows.Human recombinant Factor VIII (specific activity: 4000 IU/mg) wasobtained from Hyland (Glendale, Calif.) as material for laboratory useonly; plasma-derived (pd) Factor VIII-von Willebrand factor complex,purified by ion exchange chromatography (specific activity ±160 IU/mgprotein; 15:1 von Willebrand factor to Factor VIII w/w ratio), andpurified Factor VIII-depleted von Willebrand factor (von Willebrandfactor to Factor VIII w/w ratio 4800:1; lot 951016) were obtained fromthe Belgian Red Cross (Brussels, Belgium).

Peripheral blood samples were collected from donors suffering mildhemophilia and with inhibitors. The peripheral blood mononuclear cells(PBMC) were immortalized by EBV infection concomitantly to theactivation of surface antigens. Four hundred and eighty cell lines werescreened by ELISA for production of antibodies towards Factor VIII. Forexample, one cell line, named KRIX-1, was successfully cloned bylimiting dilution. Clonality was verified by RT-PCR amplification ofmRNA coding for the V regions of the antibody heavy and light chains: asingle sequence was obtained from 10 clones of PCR products. Purifiedantibodies were obtained by passage of KRIX-1 cell culture supernatanton Protein-A Sepharose. An ELISA performed with IgG subclass- and lightchain-specific antibodies identified KRIX-1 as an IgG4k.

Human monoclonal antibodies were purified by adsorption on immobilizedProtein A (high-TRAP^(R) Protein A; Pharmacia, Uppsala, Sweden). Fabfragments of human monoclonal antibody were prepared by papaindigestion. One mg of a selected antibody was diluted to 500 microgr/mlin phosphate buffer (40 mmol/L KH₂PO₄, 60 mM Na₂HPO₄.2H2O, 0.15M NaCl)containing 50 mmol/L L-cystein (Sigma), 1 mmol/L EDTA (Merck) and 10microgr papain (Sigma). The mixture was incubated for 3 h at 37° C. withcontinuous agitation. The reaction was stopped by addition ofiodoacetamide to a final concentration of 75 mmol/L for 30 min at RT.The digested antibody was dialysed against phosphate-buffered saline(140 mmol/L NaCl, 67 mmol/L KCl, 20 mmol/L Na₂HPO₄, 4.4 mmol/L KH₂PO₄,pH 7.4). The undigested IgG and Fc fragments were then eliminated bypassage over protein A sepharose (Hi Trap Protein A; Pharmacia). The Fabfragment was further purified by gel filtration chromatography on aSuperdex 200 (Pharmacia).

Conventional methods were used for the detection of anti-Factor VIII IgGantibodies, the determination of IgG subclass, and the evaluation ofinhibition of Factor VIII binding to von Willebrand factor. For theanalysis of the inhibition of the binding of rFactor VIII to a selectedantibody by Fab and native antibody, Maxisorb polystyrene plates (Nunc)were coated for 2 h with 50 μl of the antibody diluted to 5 microgr/mlin glycine-buffered saline (20 mmol/L glycine, 34 mmol/L NaCl, pH 9.2).After washing, 50 μl of biotin-labeled rFactor VIII diluted to 1microgr/ml in Tris-casein (10 mmol/L tris(hydroxymethyl)-aminoethane, pH7.3, containing 150 mmol/L NaCl and 0.5% casein) were mixed for 1 h at37° C. with 50 μl of human IgG at various dilutions. A 50-μl aliquot ofthe mixture was added to the plates for 2 h at RT. After washing, thebinding of biotinylated rFactor VIII was detected by sequential additionof avidin-peroxidase and OPD.

rFactor VIII (final concentration 0.2 microgr/mL) was incubated withhuman IgG antibody at different concentrations for 2 hours at 37° C. andthe residual Factor VIII activity was assessed by a chromogenic assay(Coatest® Factor VIII, Chromogenix AB, Mölndal, Sweden or Kabi Vitrum,Brussels, Belgium) Inhibition of plasma Factor VIII activity wasmeasured by the Bethesda method (Kasper et al. (1975) Thromb DiathHaemorrh 34, 612), in which a pool of normal plasma collected inbuffered trisodium citrate was used as Factor VIII source. ResidualFactor VIII activity was assessed by a chromogenic or by a one-stageclotting assay.

The Bethesda assay and residual activity measurement was performed asfollowing: one volume of antibody at various dilutions in TBS (Tris 20mM, NaCl 0.15 M, pH 7.4) was mixed with one volume of a pool of normalhuman plasma and incubated for 2 h at 37° C. The pool of normal plasmahad been constituted by mixing plasma from 10 normal individuals andbuffered by addition of Hepes (100 mM) to a final concentration of 10mM. The residual Factor VIII activity was then measured using amodification of the DADE Factor VIII chromogenic assay (Dade A G,Marburg, Germany). In this assay, thrombin-activated Factor VIIIaccelerates the conversion of Factor X into Factor Xa in the presence ofFactor IXa, PL and calcium ions; Factor Xa activity is then assessed byhydrolysis of a p-nitroanilide substrate. Reagents, which werereconstituted according to the manufacturer's instruction, comprisedbovine Factor X (1 mM), Factor IXa (0.3 mM) and thrombin (0.3 mM); CaCl₂(30 mM), PL (60 mM), a chromogenic Factor Xa substrate(CH₃OCO-D-CHG-Gly-Arg-pNA.AcOH; 3.4 mM), and a thrombin inhibitor(L-amidinophenylalanine piperidine). Aliquots of 30 μl ofplasma/antibody mixture were retrieved at the end of the 2 h incubationperiod and displayed in microtitration plates; 30 μl of the Factor X andFactor IXa/thrombin reagents were added sequentially. After 90 sec, 60μl of the chromogenic substrate was added and the incubation extendedfor 10 min at 37° C. The reaction was then blocked by addition of 30 μlcitric acid (1 M), and OD was measured at 405 nm. The residual FactorVIII activity was determined by comparing the OD₄₀₅ nm of test sampleswith that obtained with Factor VIII solutions of known concentrations.The residual Factor VIII activity was expressed as the percentage ofactivity measured in plasma aliquots handled and diluted exactly as testsamples throughout the entire experiment. Native KRIX-1 inhibited up to90% of Factor VIII activity.

Example 2 Production of Monoclonal Antibodies by Immunization in Animals

Alternatively, monoclonal antibodies having the same characteristics asdisclosed in example 1 can be produced by on purpose immunization inanimals. Thus, mice are injected with human Factor VIII in Freund'sadjuvant

Monoclonal anti-human Factor VIII antibodies are then obtained by fusionof spleen lymphocytes with a mouse myeloma cell line. Cell culturesupernatants producing anti-Factor VIII antibodies are identified andcloned by limiting dilution, using methods described in CurrentProtocols in Immunology (see supra). Further selection of inhibitorshaving the desired capacity to inhibit the pro-coagulant activity ofFactor VIII is carried out as described in example 1.

Monoclonal antibodies produced in mice are then humanized. Thus,sequences of the variable parts of mouse heavy and light chains arealigned with human immunoglobulin variable regions to identify humanantibody with the greatest homology in framework regions. The DNAfragment encoding humanized variable regions are then synthesized by aPCR-based CDR (complementarity determining regions) grafting method asdescribed for instance in Sato et al., Cancer Research (1993) 53:851-6.The final PCR product coding for the heavy chain variable part of thehumanized antibody is digested and subcloned upstream of the human Cgamma-1 gene in a first expression plasmid. The humanized light chainvariable region of the final construction is inserted upstream of the Ckappa gene in a second expression plasmid. The two constructions arethen co-transfected into COS cells expression system.

Example 3 Characterization of Anti-Factor VIII Antibodies

Monoclonal antibodies of either human (example 1) or animal (example 2)origin are characterized using an assay system by which their capacityto inhibit the binding of Factor VIII to phospholipids is evaluated.Thus, polystyrene microtitration plates are coated withphosphatidyl-L-serine. Soluble recombinant Factor VIII at 2 microgr/mlfinal concentration is mixed for 30 minutes at 37° C. with variousconcentrations of the antibody under evaluation. The mixture is thenrapidly activated with thrombin and added to phosphatidyl-L-serinecoated plates. The said plates were then incubated for two minutes at21° C. and the binding of Factor VIII was detected by addition of theanti-Factor VIII A1 domain mAbF14A2, for two minutes, followed by a twominute incubation with HRP-conjugated goat anti-mouse Fc gamma Resultsof this experiment are shown in FIG. 4 for the monoclonal antibodyproduced from the cell line KRIX-1. On the figure, the average ofactivated Factor VIII binding in the absence (closed symbols) orpresence (open symbols) of the antibody, as well as the standarddeviation of triplicates are indicated. Controls in the absence ofFactor VIII gave OD 490 lower than 0.05. FIG. 4 clearly shows that themonoclonal antibody produced from cell line KRIX-1 inhibitssignificantly the binding of Factor VIII to phospholipids but bringsabout incomplete inhibition even when added in large excess.

To demonstrate that in absence of plasma, KRIX-1 did not recognize themutated Factor VIII light chain of the donor, DNA fragments encodingwild-type and mutated Factor VIII light chains were synthesized. Thecorresponding proteins were expressed in reticulocyte lysates. Thecorrect folding of native and mutated light chains was determined byimmunoprecipitation with the human monoclonal antibody BO2C11, whichrecognizes a conformational epitope within the carboxy-terminal part ofthe Factor VIII light chain Immunoprecipitation experiments indicatedthat BO2C11 bound wild-type and Arg2150His light chains, whereas KRIX-1captured exclusively the wild-type light chain. Prolonged exposure ofSDS-PAGE gels to the autoradiography film failed to detect anysignificant binding of KRIX-1 to the mutated light chain. Controlexperiments showed no binding to assay reagents other than Factor VIIIor Factor VIII fragments, and preincubation with soluble rFactor VIIIprevented the binding to methionine-labeled Factor VIII fragments,confirming the binding specificity.

KRIX-1 did not recognize Factor VIII in Western blotting indicating thatthe epitope recognized was conformational. Further epitope mapping wastherefore performed with Factor VIII fragments produced in reticulocytelysates. Preliminary experiments had indicated that such an approach wasefficient for the synthesis of Factor VIII domains. Theimmunoprecipitation procedure using labeled Factor VIII domains producedin reticulocyte lysate was validated by mapping the epitope recognizedby the human monoclonal antibody BO2C11. A complete agreement wasobserved between the binding to Factor VIII C2 deletion fragmentsproduced in reticulocyte lysates and the binding to recombinantfragments produced in E. coli or COS cells. KRIX-1 bound to full-lengthlight chain, to fragments corresponding to A3C1, C1C2, and the isolatedC1 domain. In contrast, KRIX-1 did not bind to the C1 or C1C2 domainswith the substitution Arg2150His, although in a control experiment, theArg2150His C1C2 domain was bound by BO2C11 like its normal counterpart.

A search was made for other mutations in the light chain which couldalter the binding of KRIX-1. As shown in Table 1, KRIX-1 inhibited theactivity of all mutated Factor VIII molecules tested so far, exceptthose carrying the mutation Arg2150His.

TABLE 1 Inhibition of Factor VIII activity in mild hemophilia Apatients’ plasma. Factor VIII mutation Factor VIII activity (IU/ml)KRIX-1 Inhibition (%) Arg1689Leu 0.04 >70 Arg1749His 0.39 >70 Gly1750Arg0.38 >70 Ala1824Val 0.16 >70 Asp1825Gly 0.17 >70 His1961Tyr 0.24 >70Arg1966Gln 0.08 >70 Met2010Ile 0.04 >70 Ser2011Asn 0.23 >70 Val2016Ala0.05 >70 Asn2019ser 0.13 >70 Leu2052Phe 0.23 >70 Asp2074Gly 0.13 >70Thr2086Ile 0.08 >70 Ile2098Ser 0.20 >70 Phe2101Leu 0.06 >70 Asn2129Ser0.20 >70 Arg2150His 0.03 0 Pro2153Gln 0.02 65 Arg2159Leu 0.16 >70Trp2229Cys 0.02 >70 Gln2246Arg 0.08 >70

In the use of KRIX-1 as a medicament to partially inhibit Factor VIIImild mutations of Factor VIII will not affect the effectiveness of thetherapy.

KRIX-1 inhibited the binding of Factor VIII to von Willebrand factor ina dose-dependent manner. The concentration of KRIX-1 required to achieve50% inhibition (IC₅₀) of Factor VIII binding was 0.25 microgr/mL andmore than 95% inhibition was obtained with 20 microgr/mL of KRIX-1. Fabfragments of KRIX-1 also inhibited Factor VIII binding to von Willebrandfactor. However, on a molar basis 15 times more Fab than native KRIX-1was required to inhibit 50% of Factor VIII binding to von Willebrandfactor. Additional experiments were performed to exclude that the KRIX-1Fab fragments still contained intact or partially digested antibody.SDS-PAGE analysis of the Fab fragments purified by protein A adsorptionand gel filtration chromatography showed a single band. The presence oftrace amounts of Fc gamma fragments remaining bound to Fab fragments wasexcluded in ELISA. Wells with insolubilised Factor VIII were incubatedwith native or Fab KRIX-1. Binding of both Fab and native KRIX-1 couldbe detected by addition of peroxidase-labeled anti-kappa light chainIgG. By contrast, addition of peroxidase-labeled anti-Fc gamma IgG didnot reveal any specific binding even when 100 microgr/ml of the Fabpreparation were incubated into the wells. By comparison, the addition0.1 microgr/ml of the native antibody gave rise to a significantbinding. In ELISA, a 15-fold higher concentration of Fab than of nativeantibody was required to inhibit 50% of the binding of biotinylatedKRIX-1 onto insolubilised Factor VIII, indicating that the Fab KRIX-1fragment had a lower affinity for Factor VIII than the native antibody.Accordingly, the requirement for higher concentrations of KRIX-1 Fabthan of native antibody to inhibit Factor VIII binding to von Willebrandfactor should be attributed to the reduced affinity of KRIX-1 Fabfragments for Factor VIII.

To determine whether KRIX-1 was representative of the polyclonalantibodies from the donor, a competitive assay was used. The binding ofbiotinylated KRIX-1 to insolubilised Factor VIII was measured inpresence of increasing concentrations of either KRIX-1, polyclonal IgGfrom the donor, or control polyclonal IgG. IgG from the donordose-dependently inhibited KRIX-1 binding to Factor VIII. Theconcentration of KRIX-1 and IgG from the donor inhibiting 50% ofbiotinylated KRIX-1 on Factor VIII were of 0.3 microgr/ml and of 170microgr/ml, respectively, whereas no inhibition was observed with thecontrol IgG.

Example 4 Production of Monoclonal Antibodies Derived from Hemophilia APatients and which Bind to the Factor VIII—von Willebrand Factor Complex

Alternatively, antibodies which reduce the release rate of Factor VIIIfrom von Willebrand factor are identified as follows. Polystyrenemicrotitration plates are coated with a specific antibody to vonWillebrand factor. A solution of biotinylated recombinant Factor VIII(0.5 microgr/mL) complexed to von Willebrand factor (5 microgr/mL) ismixed with various concentrations of IgG from a donor (FIG. 5 solidsquares), e.g. the same patient as described above (from which KRIX-1was derived), MoAb4H1D7, or of IgG from a non-hemophiliac subject (FIG.5 solid triangles). IgG at the indicated concentrations was added tomicrotitration plates coated with a mouse antibody, MoAb4H1D7 againstvon Willebrand factor for an incubation of two hours at roomtemperature. After washing, Factor VIII was activated by thrombin duringtwo minutes at 37° C. Factor VIII bound to von Willebrand factor wasdetected by addition of avidine peroxidase. Controls included thedetection of bound biotinylated Factor VIII in the absence of thrombindigestion (OD450=460+47.7SD) and of biotinylated recombinant Factor VIIIafter thrombin digestion in the absence of antibody (OD450=160+16.0SD).

Results of these experiments are shown in FIG. 5 for polyclonalantibodies. On this figure, the average values as well as the standarddeviation of triplicates are indicated. FIG. 5 clearly shows that asignificantly higher proportion of activated Factor VIII remains boundto the plate in the presence of increasing concentrations of theantibody, i.e. it demonstrates a reduction of the dissociation ofactivated Factor VIII from von Willebrand factor in the presence of aninhibitor antibody recognizing Factor VIII bound to von Willebrandfactor. Monoclonal antibodies have been obtained from these polyclonalantibodies in accordance with the methods described in this invention,thus indicating that the present invention may be extended to monoclonalantibodies and fragments and derivatives thereof which bind to FactorVIII/von Willebrand factor complexes.

Example 5 Sequencing of Antibody Variable Domains

Sequencing of antibody variable domains was carried out as follows. Theisolation of RNA from EBV-immortalized human B-cell lines was performedusing TRIzol Reagent according to the manufacturer's instructions (LifeTechnologies). CDNA was synthesized with the SuperScriptpreamplification system for first-strand cDNA synthesis. The cDNAencoding the heavy chain variable region genes (V_(H)) was amplified bypolymerase chain reaction (PCR) using primers specific for the leadersequence of the V_(H) families and for the first exon of the C gammaregion, as described (Bakkus et al, Blood, 80:2326, (1992)) Annealingwas performed at 60° C. for 40 PCR cycles. PCR products of theappropriate size (460 bp) were isolated from 1.5% agarose gel and clonedusing the TA Cloning Kit (Invitrogen BV, Leek, The Netherlands). A PCRscreening using couples of primers corresponding to the V_(H) genefamily of interest was performed on cultures of randomly selectedcolonies. Plasmid DNA from positive colonies were isolated using WizardPlus Minipreps (Promega, Menlo Park, Calif.) and sequenced in bothdirections with Sequenase (US Biochemical, Cleveland, Ohio), accordingto the manufacturer's instructions. Analysis of the variable genesequences was made using the V BASE Sequence Directory (Tomlinson et al,MRC Centre for Protein Engineering, Cambridge, UK).

The complete sequences of the V_(H) and V_(L) of the antibody BO 2C11described in example 1 were submitted to the EMBL Nucleotide SequenceDatabase under the accession numbers AJ224083 and AJ224084,respectively.

The amino acid sequences shown in FIGS. 6 and 7 define the VH and VLregions of the antibody BO2C11 including the three CDR's for each of theheavy and light chains. Also given are the polynucleotide sequenceswhich encode for these regions. SEQ. ID NOs. 46 and 48 provide the aminoacid sequences of the heavy and light chains of BO2C11, respectivelywhile SEQ. ID NOs. 45 and 47 provide the polynucleotide sequences codingfor these variable regions.

The amino acid sequences shown in FIGS. 8 and 9 define the VH and VLregions of the antibody KRIX-1 including the three CDR's 1-3 for each ofthe short and long chains. Also given are the polynucleotide sequenceswhich encode for these regions. SEQ. ID NOs. 2 and 4 are the amino acidsequences of the heavy and light chains of KRIX-1, respectively whileSEQ. ID NOs. 1 and 3 provide the polynucleotide sequences coding forthese variable regions. The amino acid sequences which define the CDR1,CDR2 and CDR3 of the variable heavy chain of KRIX-1 are provided as SEQID NOs 33, 34 and 35. The amino acid sequences which define the CDR1,CDR2 and CDR3 of the variable light chain of KRIX-1 are provided as SEQID NOs 36, 37 and 38.

Example 6 (Comparative) Inhibition of Factor VIII Activity by theAntibody SAF8C-Ig

Levels of Factor VIII are measured in a functional assay following anincubation period of two hours at 37° C. with various concentrations ofthe antibody SAF8C-Ig, using the chromogenic assay described inexample 1. As shown in FIG. 10, the residual Factor VIII activity isreduced in a dose dependent manner. Already at 100 microgr/ml ofSAF8C-Ig, residual Factor VIII activity is less than 1% of the normalactivity. Such low Factor VIII levels expose the patient to a high riskof spontaneous bleedings, as is well known for instance from Levine,Ann. NY Acad. Sci. (1975) 240:201 and Gilbert, Mount Sinai J. Med.(1977) 44: 339.

Example 7 Inhibition of Venous Thrombosis in Hamsters by KRIX-1

Thrombosis was experimentally induced in the femoral vein ofanesthetized hamsters, by injecting the dye rose-bengal in the jugularvein and by exposure of the femoral vein to the green light of a Xenonlamp for 4 minutes (Kawazaki et al. Thromb Haemost (1999) 81: 306-11).As a consequence of illumination of the vessel, the dye decomposes andgenerates radicals injuring the vessel endothelial cells. Thus,subendothelial structures are exposed to the blood circulation andthrombus formation is initiated. The amount of thrombus formed ismeasured via transillumination of the injured vessel (Kawazaki et al.Thromb Haemost (1999) 81: 306-11) and is quantified via the amount ofwhite light being transilluminated through the vessel. As represented inFIG. 12, when this experiment is performed in control animals, theaverage thrombus size measured in 13 hamsters is 220,000±32,575(mean±SEM) Arbitrary light Units (A.U.), whereas treatment of a group of12 hamsters with KRIX-1 (400-800 microgr/kg, given as a bolusimmediately prior to induction of thrombosis) reduced the mean thrombussize to 122,000±27,100 A.U. (p=0.0188, Mann-Whitney test).

Additionally, the kinetics of Factor VIII inhibition by KRIX-1 wasanalyzed ex-vivo as follows: hamsters were injected intravenously withKRIX-1 (1600 microgr/kg). Levels of Factor VIII:c were measured in achromogenic assay (Coatest Factor VIII^(R) (Chromogenix A B, Mölndal,Sweden), and Factor VIII Chromogenic Assay (Dade, Düdingen,Switzerland)) using plasma collected before and at different periods oftime after injection. FIG. 11 shows that in these hamsters, Factor VIIIactivity is reduced from 1.6 IU/ml to 0.3 IU/m1 already 30 minutes afterantibody injection, thus confirming that KRIX-1 only partially inhibitsFactor VIII.

Example 8 Competition Between the Human Monoclonal Antibody RHD5 andKRIX-1

The human lymphoblastoid cell line RHD5 was derived by immortalisationof B lymphocytes from a patient who developed an autoimmune response toFactor VIII, according to described procedures (Jacquemin et al. (1998),Blood 92, 496-506). Briefly, 10⁷ peripheral blood mononuclear cells wereresuspended in 2 ml culture medium and incubated for 2 hours at 37° C.with 200 μL Epstein-Barr virus supernatant (B95-8 strain). Cells werethen seeded at 5,000 cells/well in 96-well microtiter plates (Nunc)containing feeder cells (3T6-TRAP cells irradiated with 7,000 rads). Onehundred fifty microliters of culture supernatant was replaced every weekby fresh culture medium. After 6 weeks, culture supernatant were testedin enzyme-linked immunosorbent assay for the presence of anti-FactorVIII antibodies. Positive cell lines were transferred to 24-well platesand immediately cloned at 60 cells per 96-well plate without feedercells. One clone, producing an antibody called RHD5, was selected.

This cell line producing the monoclonal antibody RHD5 was deposited withthe BCCM/LMBP (Belgian Co-ordinated Collections ofMicroorganisms/Plasmid Collection) Laboratorium voor MoleculaireBiologie, University of Ghent, Technologiepark 927, B-9052 Zwijnaarde,Belgium in August 2004, with the D. Collen Research Foundation(Onderwijs & navorsing, Campus Gasthuisberg, Herestraat 49, B-3000Leuven, Belgium) as depositor (accession number LMBP 6165CB).

The sequencing of the rearranged immunoglobulin genes coding for RHD5was performed as described in Jacquemin et al. (1998), Blood 92,496-506.

The nucleotide and amino acid sequences of the variable regions of RHD5heavy and light chain are listed in SEQ ID Nos 29 to 32. It wasdetermined that RHD5 is an IgG1.

The antibody present in the culture supernatant was purified byadsorption on HiTRAP protein A (Pharmacia).

The inhibitory activity of RHD5 was assessed in a Bethesda assay (Kasperet al. (1975), cited supra) as described in example 1. RHD5 inhibitedonly partially Factor VIII activity up to the highest concentrationtested. In a Bethesda assay performed by mixing one volume of antibodyat 200 microgram/ml or of control buffer with one volume of plasma, theresidual Factor VIII levels were 7.0±0.2 and 251.9±18.8 ng/ml,respectively (mean±SD of triplicates). The inhibition of Factor VIIIactivity reached at a final concentration of RHD5 of 100 microgram/mlwas therefore 97%. Similarly, in a Bethesda assay performed by mixingone volume of antibody at 200 microgram/ml or of control buffer with onevolume of full length recombinant Factor VIII (Recombinate^(R), Baxter),the residual Factor VIII levels were 8.0±0.2 and 399.7±18.8 ng/ml,respectively (mean±SD of triplicates). The inhibition of Factor VIIIactivity reached at a final concentration of RHD5 of 100 microgram/mlwas therefore 98%. A dose response curve of plasma Factor VIIIinhibition by RHD5 is shown in FIG. 15.

The ability of RHD5 to compete with vWF for Factor VIII binding wastested in ELISA (FIG. 16). Microtitration plates were incubatedovernight at 4° C. with the anti-vWF MoAb4H1D7 (Jacquemin et al., Blood:1998, 92: 496) diluted at 4 μg/ml. After washing the plates, 50 μl of anormal human plasma pool was added for 1 hour at RT. The plates werethen washed and then incubated for 30 minutes with 50 μl of 400 mM CaCl2to detach Factor VIII from vWF. Fifty microliters of biotinylatedrecombinant Factor VIII diluted at 0.4 μg/ml in PBS-BSA was mixed with50 μl RHD5 at different dilutions. The mixture was incubated for 30 minat 37° C. before adding a 50 μl to the plate for 2 hours at roomtemperature. After washing, bound Factor VIII was detected by additionof avidin-peroxidase followed by ortho-phenylenediamine (OPD) and theoptical density (OD) was measured at 490 nM.

The ability of KRIX-1 to compete with RHD5 for Factor VIII binding wastested in ELISA. Polystyre microtitration plates were incubatedovernight at 4° C. with 50 μL RHD5 at 2 microgram/ml in phosphatebuffered saline (PBS). The plates were washed 4 times with PBS-Tween.Biotinylated recombinant Factor VIII (0.5 microgram/ml) inTris-BSA-Tween was mixed with RHD5 or KRIX-1 at various concentrationsbefore addition to RHD5 coated plates.

After a two hours incubation period at 4° C., the plates were washed 4times and bound biotinylated Factor VIII was detected by addition ofavidine peroxidase (Sigma) at 1 microgram/ml. After 30 min at RT, theplates were washed again and supplemented with 100 μL OPD. The resultingOD was read at 490 nm in a Emax Microplate Reader (Molecular Devices,Menlo Park, Calif.).

Biotinylated Factor VIII used in the above experiment was prepared byincubating recombinant Factor VIII (100 microgram/ml) dialysed in Hepesbuffer (Hepes 10 mM, NaCl 0.15 M, CaCl2 10 mM, pH 8.5) withsulfo-NHS-LC-biotin (Pierce) at 1 microgram/ml for 2 hours at RT. Thepreparation was then dialysed against Hepes buffer and stored and −80°C.

As shown in FIG. 13, KRIX-1 was able to completely prevent Factor VIIIbinding to RHD5. This competition between KRIX-1 and RHD5 shows thatmixing the two antibodies in different ratios will allow the productionof antibody mixtures with inhibitory activity ranging between theinhibitory activity achieved with KRIX-1 (85%) and that achieved withRHD5 (97-98%).

Example 9 KRIX-1 and RHD5 Epitope Mapping

The specificity of RHD5 was further evaluated in an immunoprecipitationassay using radiolabelled recombinant Factor VIII fragments as describedin Jacquemin et al., Blood (2000), 95:156-163.

Radiolabelled native Factor VIII C1 domain and mutant C1 domain carryingthe substitution Arg2150His were incubated with human mAbs RHD5 andKRIX-1 bound to protein A sepharose. After washing, the bound materialwas eluted by boiling in SDS-buffer. The radioactivity of bound materialwas measured using a scintillation counter (A) or analysed by SDS-PAGEfollowed by autoradiography (B). Controls included a human mAb, BO2C11,directed toward the Factor VIII C2 domain and normal human plasma (H.plasma).

-   -   Expression of Factor VIII recombinant fragments in reticulocyte        transcription/translation system    -   Five hundred ng to 1 μg of DNA, linearised by Notl digestion,        was used as a template in a T7 RNA polymerase transcription        system in micrococcal nuclease-treated reticulocyte lysates        (Promega, Southampton, UK) according to the manufacturer's        instructions in the presence of L-[³⁵S]methionine (Amersham,        Bucks, UK). The [³⁵S]-methionine labeled Factor VIII fragments        migrated on SDS-PAGE as bands matching the expected mass of        corresponding Factor VIII polypeptides.    -   Immunoprecipitation of L-[³⁵S]methionine-labeled Factor VIII        fragments    -   One to three μL of standard in vitro translation product was        added to 500 μL human antibody at 2 μg/mL in NET-gel buffer (50        mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.1% Nonided NP-40; 1 mM EDTA        (pH 8); 0.25% gelatin and 5% BSA). Tubes were gently rocked for        lhour at 4° C. Twenty μL of a 50% solution of Protein A        Sepharose was then added to the antigen/antibody mixture, and        incubated for 1 hour at 4° C. on a rocking platform. The        Sepharose beads were centrifuged and washed twice with Tris-NP40        (10 mM Tris-HCl (pH 7.5); 0.1% NP40). Bound antigen/antibody        complexes were eluted from the beads by boiling for 4 minutes in        30 μL of SDS gel loading buffer. The radioactivity of eluted        material was measured using a scintillation counter. In        addition, an aliquot of 15 μL was analysed by 10% (w/v)        polyacrylamide gel electrophoresis and visualized by        autoradiography. Control experiments were performed with the        human monoclonal antibody BO2C11, directed towards the Factor        VIII C2 domain (Jacquemin et al., Blood (1998), 92: 496-506) and        normal donor's polyclonal IgG antibodies purified on Protein A        Sepharose.

KRIX-1 and RHD5 bound the isolated C1 domain (FIG. 17). By contrast, asshown in FIG. 17, neither KRIX-1 (LE2E9) nor RHD5 bound to the C1 domainwith the substitution Arg2150His. In a control experiment, the C1 domainwas not captured by the mAb BO2C11 recognising the C2 domain (Jacqueminet al. (1998) 92: 496-506) nor by control polyclonal antibodies.

Example 10 Effect of RHD5 on Bleeding After Tail Cutting in Wild-TypeMice

The risk of severe bleeding associated with high concentrations ofmAb-RHD5 in plasma was evaluated in a tail cutting experiment. Thisassay is based on the observation that section of the distal portion ofthe tail results in important blood loss leading to death in most FactorVIII deficient mice (Bi et al. Nat Genet. (1995) 10:119-2) whereasnormal mice or animals with low Factor VIII levels survive. Thisprocedure allows an in vivo evaluation of Factor VIII activity.

In a preliminary experiment, we compared Factor VIII inhibition bymAb-RHD5 in human and mice plasma. One volume of mice plasma or of humanplasma was mixed with one volume of mAb-RHD5 at 10 μg/ml in 0.15 M NaCl,0.5% bovine serum albumin, 50 mM Tris(hydroxymethyl)-aminomethane, pH7.2. After a 2 h incubation period at 37° C., the residual Factor VIIIactivity was measured with a Factor VIII chromogenic assay (DadeBehring, Marburg, Germany) according to the manufacturerrecommendations. Mab-RHD5 inhibited 80% Factor VIII activity in humanplasma but did not inhibit Factor VIII activity in mice plasma. Bycontrast, mAb-RHD5 inhibited recombinant human Factor VIII added toplasma of Factor VIII deficient mice. Because mAb-LE2E9Q did not inhibitmice Factor VIII, a tail clipping experiment could not be performed inwild type mice. The experiment was therefore performed in Factor VIIIdeficient mice in which normal Factor VIII levels had been obtained byadministration of recombinant human Factor VIII.

Factor VIII deficient mice (n=6) were injected intravenously with 10 IUrecombinant human Factor VIII and 10 min later with 100 μg mAb-RHD5. A7-mm section of the tail was cut 30 minutes later and survival ratemonitored over the subsequent 24 hours. A group of 6 Factor VIIIdeficient mice was used as control. After 24 h, 5 mice were dead in thecontrol groups whereas all animals that had received recombinant humanFactor VIII followed by mAb-RHD5 administration survived. Thisexperiment demonstrated that in vivo RHD5 only partially inhibits theFactor VIII, even when the antibody is in large excess (100 μg antibodyfor about 2 μg recombinant human Factor VIII).

Example 11 Identification of Alternative Inhibitory Antibodies to FactorVIII

The present example describes how, starting from a first inhibitoryantibody such as KRIX-1, additional partially inhibitory antibodies canbe identified, based on the fact that they compete with binding of afirst inhibitory antibody such as KRIX-1 to Factor VIII. The proceduredescribed below can be performed similarly using RHD5 in stead of KRIX-1as a first inhibitory antibody. Similarly, the first inhibitory antibodyused in these assays can be a glycosylated, partially glycosylated orcompletely degylosylated (extensive enzymatic treatment or site directedmutagenesis at essential positions in the glycosylation consensussequence) of RHD5 or KRIX-1, provided that said antibody is stillcapable of binding to KRIX-1 and of partially inhibiting the activity ofFactor VIII.

An example of an assay to identify further inhibitory antibodies is onewherein labelled KRIX-1 (radioactive labelled or labelled with biotin orwith a chromophoric group) is bound to Factor VIII. Uncharacterisedantibodies are then screened for their ability to disrupt the binding ofKRIX-1 to Factor VIII. A large number of uncharacterized antibodies canbe screened simultaneously.

Alternatively the uncharacterised antibodies are first incubated withFactor VIII insolubilised on microtiter plates, whereafter labelledKRIX-1 is added and assayed for its binding to Factor VIII.Alternatively, KRIX-1 and the uncharacterized antibody are mixedtogether before assaying the residual binding of KRIX-1 to Factor VIII.

Using these assays, antibodies which impair the binding of KRIX-1 to theC1 domain of Factor VIII can be identified. This impairment can beachieved by an antibody directed to the same epitope in the C1 domain asfor KRIX-1, by an antibody directed to another epitope that the one ofKRIX-1 in the C1 domain, or by antibody with an epitope outside the C1domain but which sterically competes with the binding of KRIX-1 antibodyto its epitope in the C1 domain.

The screening for antibodies can for example be initiated by screeningin first instance a scFv library for scFv fragments that bind to humanFactor VIII and more particularly bind to the C1 domain of Factor VIII.For this technique, antibody fragments have been displayed on thesurface of filamentous phage that encode the antibody genes (Hoogenboomand Winter (1992) J Mol Biol. 227, 381-388; Vaughan et al. (1996) Nat.Biotechnol. 14, 309-314; Tomlinson et al. (1992) Hum Mol Genet. 3,853-860; Nissim et al. (1994) EMBO J 13, 692-698; Griffiths et al.(1994) EMBO J. 12,725-734). Variable heavy chain (VH) and variable lightchain (VL) immunoglobulin libraries can be developed in phages. Thesephages can then be selected by panning with antigen (Factor VIII, or theC1 domain of Factor VIII). The encoded antibody fragments can then besecreted as soluble fragments from infected bacteria. This display ofantibodies on phages and the selection with antigen mimics immuneselection and can be used to make antibodies without immunizationstarting from a single library of phages (Hoogenboom and Winter (1992) JMol Biol. 227, 381-388). Alternatively, the phages can be selected bypanning with the first inhibitory antibody. A human synthetic VH andVscFv library has been made by recloning the heavy and light chainvariable regions from the lox library vectors, wherein the heavy andlight chain V-genes were shuffled at random and cloned for display assingle-chain Fv (scFv) fragments on the surface of filamentous phage(Griffiths et al. (1994) EMBO J. 12,725-34) [Centre for ProteinEngineering of Dr. G. Winter, LMB-MRC, Cambridge, UK] into the phagemidvector pHEN2.

Depending on the selection criteria used in the first step, antibodyfragments can be identified which bind to the C1 domain of Factor VIIIor which compete with the binding of an antibody such as KRIX-1 toFactor VIII. Hereafter, these fragments can be screened for ability tocompete with the binding of a first inhibitory antibody or theiraffinity of binding to Factor VIII, respectively. Subsequently, they canbe tested for their ability to inhibit Factor VIII activity and for thepresence of a plateau effect of Factor VIII inhibition at a molarexcess.

Considering the size of the fragments, it is envisaged that enlargingthe size of these fragments, by cloning these scFv fragments into acomplete antibody, will result in an increased inhibitory activity.

Alternatively, the identification of further inhibitory antibodies isdone by screening antibodies isolated from one or more hemophiliapatient(s) or by screening antibodies obtained by traditionalimmunization with Factor VIII or a fragment thereof comprising the C1domain. These antibodies can also be tested in the competition assaydescribed above.

Antibodies or fragments which have been obtained using the abovementioned assay are tested for their inhibitory effect on Factor VIIIactivity and/or for their capacity to disrupt a complex between FactorVIII and e.g. vWF. Further, inhibitory antibodies or fragments are thenscreened for the presence of partial Factor VIII inhibition atphysiological excess (“plateau effect”).

Further to obtaining these alternative inhibitory antibodies and/orantibody fragments it can be envisaged to modify the glycosylation ofthe antibodies in accordance with the invention.

Any second antibody which competes with KRIX-1 binding can be used inmixtures together with at least one other antibody which competes withthe binding of native KRIX-1, including KRIX-1 itself or a fragment ofnative KRIX-1, or a modified version of KRIX-1 or a fragment thereof,more particularly a KRIX-1 or fragment thereof with a modifiedglycosylation, which mixtures have a resulting Factor VIII inhibitoryactivity which is an intermediate between the inhibitory activity ofeach of the at least two antibodies in the mixture (see also example 14below). Alternatively, the mixtures with intermediate partial inhibitoryactivity can be obtained by combining at least two antibodies orfragments thereof capable of binding to the C1 domain and capable ofcompeting with the binding of antibody RHD5 to Factor VIII.

Example 12 Effect of Deglycosylation on Factor VIII Inhibition by KRIX-1

KRIX-1 (0.5mg/m1 in PBS) was mixed with N-glycosidase-F (rochediagnostics Gmbh, Mannheim, Germany) at final concentration of 2 U/ml.The mixture was incubated at 37° C. during 72 hours under gentlestirring.

The inhibitory activity of native and deglycosylated KRIX-1 was assessedin a Bethesda assay (Kasper et al. (1975), cited supra). Therefore, onevolume of antibody at various dilutions in TBS (Tris 20 mM, NaCl 0.15 M,pH 7.4) was mixed with one volume of a pool of normal human plasma andincubated for 2 h at 37° C. The pool of normal plasma had beenconstituted by mixing plasma from 10 normal individual and buffered byaddition of Hepes (100 mM) to a final concentration of 10 mM. Theresidual Factor VIII activity was then measured using a modification ofthe DADE Factor VIII chromogenic assay (Dade A G, Marburg, Germany). Inthis assay, thrombin-activated Factor VIII accelerates the conversion ofFactor X into Factor Xa in the presence of Factor IXa, PL and calciumions; Factor Xa activity is then assessed by hydrolysis of ap-nitroanilide substrate. Reagents, which were reconstituted accordingto the manufacturer's instruction, comprised bovine Factor X (1 mM),Factor IXa (0.3 mM) and thrombin (0.3 mM); CaCl₂ (30 mM), PL (60 mM), achromogenic Factor Xa substrate (CH₃OCO-D-CHG-Gly-Arg-pNA.AcOH; 3.4 mM),and a thrombin inhibitor (L-amidinophenylalanine piperidine). Aliquotsof 30 μl of plasma/antibody mixture were retrieved at the end of the 2 hincubation period and displayed in microtitration plates; 30 μl of theFactor X and Factor IXa/thrombin reagents were added sequentially. After90 sec, 60 μl of the chromogenic substrate were added and the incubationextended for 10 min at 37° C. The reaction was then blocked by additionof 30 μl citric acid (1 M), and OD was measured at 405 nm. The residualFactor VIII activity was determined by comparing the OD_(405 nm) of testsamples with that obtained with Factor VIII solutions of knownconcentrations. The residual Factor VIII activity was expressed as thepercentage of activity measured in plasma aliquots handled and dilutedexactly as test samples throughout the entire experiment.

Native KRIX-1 inhibited up to 90% of Factor VIII activity. By contrast,a maximal inhibition (plateau inhibition) of only 50% was achieved withdeglycosylated KRIX-1 (FIG. 18).

Example 13 Mixing Native and Deglycosylated KRIX-1 allows the Selectionof Antibody Mixtures Inhibiting Factor VIII to Different Levels

Mixtures containing different ratios of KRIX-1 deglycosylated withN-glycosidase-F versus native KRIX-1 were prepared. Each mixture wasdiluted to various antibody concentrations ranging between 0.05 and 25microgram/ml. One volume of each dilution was mixed with one volume of apool of normal human plasma. After a 2 hour incubation at 37° C., theresidual Factor VIII was assessed using a chromogenic assay (Factor VIIIChromogenic assay, Dade Behring, Marburg, Germany). The native anddeglycosylated KRIX-1 inhibited Factor VIII activity by about 90% and50%, respectively (FIG. 19). By contrast, a mixture of 4.5 nativeantibody for 1 deglycosylated antibody resulted in a maximal Factor VIIIinhibition or plateau inhibition of about 80% whereas a mixturecontaining 1.5 native KRIX-1 for 1 native antibody inhibited about 65%Factor VIII activity (FIG. 19). Mixtures inhibiting Factor VIII activityto any level comprised between 50 and 90% can be similarly obtained byvarying the ratio of native and deglycosylated KRIX-1.

Example 14 Recombinant KRIX-1 Produced in CHO Cells (CHO-recKRIX-1) hasa Lower Factor VIII Inhibitory Activity than KRIX-1 (Produced by a HumanLymphoblastoid Cell Line)

RNA from KRIX-1 EBV-immortalised human B cells was isolated using TRIzolReagent according to the manufacturer's instructions (LifeTechnologies). cDNA was synthesised with the SuperScriptpre-amplification system for first-strand cDNA synthesis.

The sequences encoding the heavy or light chain were amplified by RT.PCRon mRNA prepared from KRIX-1 cells using the QuickPrep®Micro mRNAPurification Kit (Amersham Pharmacia Biotech, Rosendaal, TheNetherlands). Specific PCR primers for the heavy chain were: forwardprimer 5′-cggggtaccccaccATGGACTGGACCTGGAGGATC-3′ (SEQ ID NO:5)corresponding to nucleotides (nt) 1 to 21 (in capitals) of the cDNAsequence (WO 01/04269 A1), and containing a KpnI site (underlined) forcloning purposes and a Kozak sequence (bold italic); reverse primer:5′-tatggccgacgtcgactcATTTACCCGGAGACAGGGAGAG-3′ (SEQ ID NO: 6)corresponding to nt 1800-1780 (capitals) of the 3′ end of the humangamma-4 constant region (accession number K01316) and containing a stopcodon (bold italic) and a SalI site (underlined) for cloning purposes.Specific primers for the light chain were: forward primer5′-cccaagcttccaccATGGAAACCCCAGCKCAGCT-3′ (SEQ ID NO: 7) corresponding tont 1-20 (capitals) of the cDNA sequence (WO 01/04269 A1), and containinga HindIII site (underlined) for cloning purposes and a Kozak sequence(bold italic); reverse primer:5′-aaacagcctctagactaACACTCT-CCCCTGTTGAAG-3′ (SEQ ID NO: 8) correspondingnt 653-635 of the 3′ end of the human kappa constant region (accessionnumber V00557) and containing a stop codon (bold italic) and a XbaI site(underlined) for cloning purposes. After sequence verification, theheavy and light chain sequences were cloned consecutively into thepBudCE4 plasmid (Invitrogen, Merelbeke, Belgium) designed for doublegene expression in eukaryotic cells under the control of the EF1-alphaand the CMV promoter, respectively, using the above indicatedrestriction sites. The final vector was used for stable transfection ofCKO-K1 cells using the FuGENE6 system (Roche Diagnostics, Brussels,Belgium) according to the manufacturer's instructions. The transfectedcells were cultured in DMEM (Life Technologies, Paisley, UK)supplemented with 10% FCS, 4 mmol/L glutamine and 80 mg/L gentamicine(Geomycin®, Schering-Plough, Heist-op-den-Berg, Belgium) in the presenceof zeocin (0.7 mg/mL selection concentration or 0.35 mg/mL maintenanceconcentration; Life Technologies, Invitrogen), and were verified forantibody production by ELISA (see below). The cells were adapted togrowth in serum-free medium by step-wise reduction of the FCS to 0%, andafter clonal dilution, the best producer in terms of functionality(ELISA on huFactor VIII), as well as expression (ELISA withanti-humanlgG4 detection antibody), was used for batch production.

For detection of anti-Factor VIII antibodies, rFactor VIII wasinsolubilised by incubating plates for 2 h at 4° C. directly with 50 μlof rFactor VIII (1 microgram/ml) diluted in glycin-buffered saline(GBS). The plates were washed as above and 50 μl of culture supernatantwere added for a further incubation of 2h at 4° C. After washing, 50 μlperoxidase-labelled anti-human Fc gamma goat IgG (Sigma) diluted1000-fold in Tris-casein were added. After 2 h at RT, the plates werewashed again and supplemented with 100 μl OPD. The resulting OD was readat 492 nm in a Emax Microplate Reader (Molecular Devices, Menlo Park,Calif.). Negative and positive controls were culture medium and IgGpurified from a high-titer inhibitor hemophilia A patient, respectively.

The recombinant antibody was purified from the cell culture supernatantby adsorption on immobilized protein A (High-TRAP Protein A, Pharmacia,Uppsala, Sweden). Culture supernatant was passed through a high-TRAP®protein A (Pharmacia, Uppsala, Sweden) at a flow rate of 1 ml/min. BoundIgG was eluted with citric acid 100 mM, pH3. After pH neutralisationwith Tris pH9, IgG was dialysed against Phosphate buffered saline (PBS).The concentration of proteins was determined with the Bio-Rad assay(Biorad).

The recombinant antibody produced in CHO cells was called CHO-recKRIX-1.Interestingly, the maximal inhibition observed in large excess of thisantibody reaches only 75-85% Factor VIII activity, which is lower thanthe 85-95% maximal (plateau) inhibition observed when Factor VIII isincubated with KRIX-1 (produced by the human lymphoblastoid cell line(FIG. 20).

Example 15 Prevention of Vena Cava Thrombosis Using CHO-recKRIX-1 inMice

Thrombus was produced in the inferior vena cava of adult male wild-typemice (weight 18g-31g, age 8-10 weeks) using a previously described model(Singh et al. (2002) cited supra). Mice were anaesthetised withisoflurane, the inferior vena cava was exposed below the renal veins viaa median laparotomy and a neurosurgical vascular clip (Braun Medical)was applied for 15 seconds on two occasions, 30 seconds apart to asegment of the vena cava. A 5/0 prolene thread was then placed alongsidethe vena cava and a stenosis produced by tying a 4/0 silk suture aroundthe vena cava and the prolene thread. The thread was removed to allowblood flow to resume. The abdomen was closed and the animal allowed torecover. After 4 hours, the mice were reanaesthetised and a 1 cm portionof the inferior vena cava (between the point of ligature and iliacbifurcation) was excised and examined for the presence of thrombus. Theexcised segments were then washed in 10% PBS and soaked overnight in 1%paraformaldehyde. Vessel segments were embedded in paraffin wax and 7×10μm transverse sections were cut at 0.5 mm intervals from the ligaturedown.

Sections were stained by haematoxylin and eosin, Martius Scarlet Blue(MSB) and a rabbit anti-platelet antibody (Accurate Chemical &Scientific Corporation, Westbury, N.Y. 11590). MSB stains fresh fibrinred or mature fibrin blue/gray, red cells yellow and collagen brightblue. Thrombus size was measured by scoring the 7 sections for thepresence of thrombus, giving a score of 1 for the presence and 0 for theabsence of thrombus in each. Scores were then added up for each animal.The investigators performing the operations and the microscopic analyseswere blinded towards treatment groups.

Thrombosis was induced in three groups of wild-type mice 16 hours aftersubcutaneous injection of 150 microgram of antibody or saline. Thestatistical significance of differences between groups was evaluated onthe presence or absence of thrombus using Fisher's exact test (2-sided).The effects on thrombus size were tested by comparing thrombus scoresusing the Mann-Whitney U test.

Ten out of 14 mice injected with saline developed a thrombus, visiblemacroscopically, compared with 0 out of the 7 animals in each of thegroups pretreated with either KRIX-1 or CHO-recKRIX-1 (P<0.01).

Histological analysis identified thrombi in 11 out of 14 control animalsand 1, 1, and 2 thrombi, respectively, in animals treated with KRIX-1 orCHO-recKRIX-1 (FIG. 21). Accordingly, although CHO-recKRIX-1 inhibitsFactor VIII activity significantly less than KRIX-1, CHO-recKRIX-1inhibits very efficiently thrombosis and therefore offers a bettersafety/efficacy profile than the native KRIX-1 antibody.

Example 16 Antithrombotic Activity of CHO-recKRIX-1 in Mice with Type IIHeparin Binding Site (HBS) Antithrombin Deficiency (AT^(m/m)))

The antithrombotic efficacy of CHO-recKRIX-1 was evaluated using thethrombotic priapism model in mice with type II heparin binding site(HBS) antithrombin deficiency (Dewerchin et al. submitted).

The mice were previously generated by targeted knock-in of an R48Cmutation (corresponding to the “Toyama” R47C mutation in man, abolishingheparin/heparan sulphate binding and cofactor activity (Koide et al.(1983) Thromb Res. 31, 319-328; Koide et al. (1984) Proc Natl Acad SciUSA. 81, 289-293) in the HBS of antithrombin (AT) (AT^(m/m) mice),resulting in life-threatening, spontaneous thrombosis at differentsites, most prominently in the heart, liver, and in ocular, placentaland penile vessels (Dewerchin et al. (2003) Circ Res 93,1120-1126). Theobservation of priapism occurrence upon mating of males AT^(m/m)provided the basis to the development of a physiological model of venousthrombosis, providing a defined endpoint and an easy grading of thethrombotic outcome.

Age-matched groups of sexually mature males (2 to 4 months) weresubcutaneously injected twice (three days before mating and on the dayof mating) with 100 μl of saline or with 100 μl of a 1 mg/ml solution ofKRIX-1, CHO-recKRIX-1Q or CHO-recKRIX-1. After the second injection,each male was mated to two wild type Swiss females, which were replacedby two new females on day 3 after mating. The formation of a vaginalmucus plug indicating recent mating was recorded daily for all females,and only the results obtained with males with confirmed sexual activitywere incorporated in the analysis. Males were examined daily fordevelopment of priapism and were sacrificed when priapism was observed,or at day 8 after initial mating when the experiment was ended. Atsacrifice, blood samples were collected for determination of residualFactor VIII activity and human IgG levels as described above. Thepenises were dissected and the presence of thrombus IN the dorsal penilevein and corpora cavernosae determined by visual inspection.

After sacrifice, the dissected penises were paraformaldehyde fixed,parafin-embedded and processed for histological analysis. Seven-μmtransverse sections were stained with haematoxylin/eosin for microscopicanalysis.

Scoring: Thrombotic outcome was scored using four categories: 0, nothrombosis; 1, thrombosis of the penile vein by microscopy; 2,macroscopically visible thrombosis of the penile vein; 3, irreversiblethrombotic priapism. When no macroscopically visible thrombus wasobserved and no histology of the penile vein could be obtained fortechnical reasons, the animals were also scored 1. The investigatorsperforming the injections and monitoring the mice were blinded towardsthe treatment groups. The statistical significance of differencesbetween thrombus scores was tested using the Kruskal-Wallis orMann-Whitney U test.

The presence of a vaginal mucus plug in at least 2 females within thefollow-up period for each these males treated with antibody or saline,confirmed actual sexual activity of the males.

KRIX-1, CHO-rec-KRIX-1 were able to prevent priapism in all mice tested(p<0.05 versus saline) (FIG. 22). In the group injected with 2×100 μgKRIX-1 antibody, none of the five males developed priapism; four of themwere also free of thrombosis upon visual inspection and by microscopicanalysis at the end of the experiment; the remaining male did not showmacroscopic thrombosis. For technical reasons, no histological analysiscould be performed and the animal was therefore scored 1 (FIG. 22), themaximal score which could have been attributed if the analysis had beenperformed.

A similar outcome was observed for the recombinant CHO-rec-KRIX-1antibody: none of seven treated males developed priapism; five maleswere also free of macroscopic or microscopic thrombosis (FIG. 22); onemale showed only microscopically detectable thrombosis (score 1) (FIG.22) and one male was free of macroscopically visible thrombosis butcould not be analyzed by microscopy and was therefore also scored 1(FIG. 22).

Example 17 Antithrombotic Activity of CHO-recKRIX-1Q in Mice with TypeII Heparin Binding Site (HBS) Antithrombin Deficiency (AT^(m/m))

As outlined in example 16, the antithrombotic efficacy of CHO-recKRIX-1Qwas evaluated using the thrombotic priapism model in mice with type IIheparin binding site (HBS) antithrombin deficiency (Dewerchin et al.(2003) Circ Res 93,1120-1126).

In the present example, age-matched groups of sexually mature males weresubcutaneously injected twice (three days before mating and on the dayof mating) with 100 μl of saline or with 100 μl of a 1 mg/ml solution ofCHO-recKRIX-1Q, a control human IgG4 monoclonal antibody, which does notrecognise Factor VIII, or the vehicle (PBS).

CHO-recKRIX-1 was able to reduce thrombosis development (p<0.05 versusPBS and control IgG4) (FIG. 27). In the group injected with 2×100microgram CHO-recKRIX-1Q antibody, none of the males died or developedpriapism. All animals treated with CHO-recKRIX-1Q were also free ofthrombosis upon visual inspection For technical reasons, no histologicalanalysis could be performed and the animal were therefore scored 1 (FIG.27), the maximal score which could have been attributed if the analysishad been performed. By contrast, in the groups treated with PBS or acontrol human IgG4 monoclonal antibody, several animals died ordeveloped priapism (p<0.01, CHO-recKRIX-1Q versus PBS and control IgG4).

Example 18 Production and Characterisation of Variant of CHO-recKRIX-1Devoid of N-glycosylation Site in the Antigen Binding Site

CHOrecKRIX-1Q was produced by site directed mutagenesis on thepCR4-Blunt-TOPO-KRIX-1H plasmid resulting in a single amino acid changein the heavy chain altering the Asn47 into Gln47 in order to disrupt theN-linked glycosylation site at Asn47-Thr49. Other plasmids comprisingthe coding sequence of the KRIX-1 antibody can similarly be used in thecontext of the present invention. Amino acid sequences comprising theCDRs of the heavy and light chains of KRIX-1 are provided in SEQ IDNOs33 to 35 and 36 to 38 for the heavy and light chains, respectively.Nucleotide sequences encoding sequences of the CDRs of the heavy andlight chains of KRIX-1 can be identified within the nucleotide sequenceencoding the heavy and light chain variable region of KRIX-1 provided inSEQ ID NO:1 and SEQ ID NO: 3, respectively.

The mutagenesis at Asn47 was obtained using the Site DirectedMutagenesis Kit (Stratagene, La Jolla, Calif.) in combination with thefollowing specific PCR primers:

Forward Primer:

5′-CCTGCAAGACCTCTGGATACcAaTTCACCGGCTACTCTGCTTCTGG-3′ (SEQ ID NO: 9)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (a to c and c to a; bolditalic);

Reverse Primer:

5′-CCAGAAGCAGAGTAGCCGGTGAAtTgGTATCCAGAGGTCTTGCAG-G-3′ (SEQ ID NO: 10)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (g to t and t to g; bolditalic)

CHO-recKRIX-1A was produced by site directed mutagenesis resulting in asingle amino acid change altering Thr49 into Ala49 in order to disruptthe N-linked glycosylation site at Asn47-Thr49

This was obtained using the Site Directed Mutagenesis Kit (Stratagene,La Jolla, Calif.) in combination with the following specific PCRprimers:

Forward Primer:

5′-CCTCTGGATACAACTTCgCtGGCTACTCTGCTTCTGG-3′ (SEQ ID NO: 11)corresponding to nt 128 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (a to g and c to t; bolditalic);

Reverse Primer:

5′-CCAGAAGCAGAGTAGCCaGcGAAGTTGTATCCAGAGG-3′ (SEQ ID NO: 12)corresponding to nt 128 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (g to a and t to c; bolditalic);

CHO-recKRIX-1E was produced by site directed mutagenesis resulting in asingle amino acid change altering Asn47 into G1u47 in order to disruptthe N-linked glycosylation site at Asn47-Thr49

Forward Primer:

5′-CCTGCAAGACCTCTGGATACgAgTTCACCGGCTACTCTGCTTCTGG-3′ (SEQ ID NO: 13)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (a to g and c to g; bolditalic);

Reverse Primer:

5′-CCAGAAGCAGAGTAGCCGGTGAAcTcGTATCCAGAGGTCTTGCAG-G-3′ (SEQ ID NO: 14)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing two altered nucleotides (g to c and t to c; bolditalic).

CHO-recKRIX-1D was produced by site directed mutagenesis resulting in asingle amino acid change altering Asn47 into Asp47 in order to disruptthe N-linked glycosylation site at Asn47-Thr49.

Forward Primer:

5′-CCTGCAAGACCTCTGGATACgACTTCACCGGCTACTCTGCTTCTGG-3′ (SEQ ID NO: 15)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing one altered nucleotide (a to g; bold italic);

Reverse Primer:

5′-CCAGAAGCAGAGTAGCCGGTGAAGTcGTATCCAGAGGTCTTGCAG-G-3′ (SEQ ID NO: 16)corresponding to nt 119 to 164 of the KRIX-1 Heavy chain sequence(capital) containing one altered nucleotide (t to c; bold italic)

After sequence verification, the mutated heavy and wild-type (native)KRIX-1 light chains were cloned into the pEE6.4 and pEE14.4 vector(Lonza Biologics, Portsmouth, N.H.) respectively. The two vectors werecombined to a double gene vector—containing both heavy and lightchain—using the Nod and SalI restriction sites present in both vectors.Heavy and light chain expression in eukaryotic cells is under thecontrol of the hCMV-MIE promoter (present in pEE14.4 and pEE6.4). Thedouble gene vector was linearised with SalI before transfection.

The linearised vector was used for stable transfection of CKO-K1 cellsusing the FuGENE6 transfection reagent (Roche, Brussels, Belgium)according to the manufacturer's instructions. The transfected cells werecultured in glutamine-free DMEM (JRH Biosciences, Lenexa, Kans.)supplemented with FBS 10%, GS Supplement (JRH Biosciences, Lenexa,Kans.) and 25 μM L-Methionine Sulfoximine (MSX) (Sigma-Aldrich, Bornem,Belgium) for selection.

The best producers were adapted to growth in serum-free medium (EX-CELL302 serum-free medium w/o L-Glutamine, JRH Biosciences, Lenexa,Kans.)—supplemented with 25 μM MSX and GS Supplement—by step-wisereduction of the FBS to 0%. The best expressing (ELISA withanti-humanIgG4 detection antibody) functional cell line was used forbatch production of the mutated rec-mAb-KRIX-1, either using theadherent or the suspension cell line.

The recombinant antibody was purified from the cell culture supernatantby affinity chromatography using a HiTrap rProtein A FF column (AmershamBiosciences, Uppsala, Sweden). After concentration the rec-mAb-KRIX-1Q(A, E and D resp.) were assayed for functionality (Chromogenic assay toevaluate the ability of the mutated rec-mAb KRIX-1 to inhibit fVIIIactivity) Inhibitory capacity towards fVIII was compared to that of thewild type rec-mAb KRIX-1 (FIGS. 23 and 24). Factor VIII inhibition bythese mutants ranged from 30 to 40%.

Measurement of Surface Plasmon Resonance (SPR).

The rate of Factor VIII association and dissociation to CHO-rec-KRIX-1Q,CHO-rec-KRIX-1A and native CHO-rec-KRIX-1 was analysed using a PharmaciaBiosensor BIAcore™ instrument (Pharmacia Biosensor AB). Purifiedantibody (20 microgram/ml in 10 mM sodium acetate buffer pH 5.0) wasimmobilised on the activated surface of a CM5 sensor chip, according tothe manufacturer's instructions. All binding experiments were carriedout in HBS at a constant flow rate of 10 μl/min. Factor VIII in HepesBuffered Saline (HBS) was infused at various concentrations over theantibody coated on the sensor chip surface. At the end of each cycle,the surface was regenerated by flushing HCl, pH 2, for 36 sec. Controlexperiments ensured that Factor VIII bound only to insolubilisedantibody. Thus, rFactor VIII did not bind to the sensor chip in theabsence of antibody, and preincubation of rFactor VIII with solubleantibody prior to addition to the chip completely prevented Factor VIIIbinding.

Association and dissociation rate constants were determined bynon-linear fitting of individual sensorgram data (O'Shanessy et al.(1993), Analyt Biochem 212: 457) using the BIA evaluation 2.1 software(Pharmacia Biotech, Uppsala, Sweden). Values of k_(ass) and k_(diss)were determined by averaging the values obtained for individual curvesestablished with various analyte concentrations. Values of k_(diss) weredetermined from the individual curves obtained with only the highestanalyte concentration, in order to reduce bias due to rebinding of theanalyte to free immobilized ligand. All data were analysed aftercorrection of the baseline by subtracting the response observed beforeinjection of the analyte (rfVIII) from the response values obtainedduring the association and dissociation phases.

The dissociation constant (K_(D)) of Factor VIII from CHO-rec-KRIX-1Q,CHO-rec-KRIX-1A and native CHO-rec-KRIX-1 was very similar (Table 2).Accordingly, the glycosylation site in the antigen binding site of mAbKRIX-1 influences the antibody inhibitory activity but does notcontribute significantly to binding to Factor VIII.

TABLE 2 surface plasmon resonance analysis of Factor VIII binding to mAbKRIX-1 and derivative thereof. Modified mAb KRIX-1 (LCL): K_(D) (nM)CHO-recKRIX-1 0.14 ± 0.03 CHO-recKRIX-1Q 0.17 ± 0.02 CHO-recKRIX-1A 0.13± 0.01

Example 19 Prevention of Arterial and Venous Thrombosis in BaboonsMethods

Protocol

Male baboons (Papio ursinus) were used. The animals weighed between 8and 17 kg and were disease-free for at least 6 months prior to theexperiments. All procedures were approved by the Ethics Committee forAnimal Experimentation of the University of the Free State in accordancewith the National Code for Animal Use in Research, Education, Diagnosisand Testing of Drugs and Related Substances in South Africa.

Permanent polytetrafluoroethylene (Teflon) and silicone rubber(Silastic) arteriovenous (AV) shunts were implanted in the baboonfemoral vessels. Blood flow through the shunts varied between 100 and120 mL/min. Handling of the baboons was achieved through anesthesia withketamine hydrochloride (Anaket-V, Centaur Laboratory).

In each experiment, a thrombogenic device prefilled with saline to avoida blood-air interface was incorporated as an extension segment into thepermanent arteriovenous shunt by means of Teflon connectors (Kotze etal. (1983) Thromb Haemost. 70, 672-675). Platelet-dependent arterialthrombus was induced by using Dacron inserted into the wall of Silastictubing (3-mm inside diameter) according to Hanson et al. (1985)Arteriosclerosis 5, 595-603 (FIG. 25).

The Dacron vascular graft material (1.26 cm²) served as a generator ofplatelet-dependent arterial-type thrombosis. An expansion chamber (3.77cm²) was used to generate coagulation-dependent venous thrombosis. Bloodflowed through the thrombogenic devices at a rate of approximately 120ml/min. The initial shear stress was 318 sec⁻¹ for the Dacron sectionand 10 sec⁻¹ for the expansion chamber.

In the control studies, the devices were kept in place for 60 minutes oruntil they occluded, after which they were removed and blood flowthrough the permanent AV-shunt re-established. The baboons were thentreated with a single intravenous bolus of 1.25 or 5 mg/kgCHO-rec-KRIX-1Q. The thrombogenic devices were placed for 60 minutes, 1h after antibody injection, after which the devices were removed andblood flow through the permanent AV-shunt reestablished. Additional60-minute studies were carried at 24 h and 48 h after the antibody bolusinjection. The extracorporeal shunts were then removed after the lastthrombosis experiment. Blood samples were taken according to thesampling schedule either directly from the shunt or by venopuncture.Factor VIII activity, mAb KRIX-1 concentrations monitored, PT, APTT,fibrinogen, were measured on all samples.

Graft Imaging.

Autologous platelets were labeled with 111In-tropolone and reinjectedinto the animal 1 h before the start of the control experiment. Thisallowed image acquisition on day 0, 1 and 2. To provide imageacquisition on day 6 or 14 the labeling procedure was repeated. Imageacquisition of the grafts was done with a gamma scintillation camerafitted with a high-resolution collimator. The images were stored on andanalysed with a computer imaging and analysis system interfaced with thescintillation camera. Dynamic image acquisition, 3-minute image of a 5m1autologous blood sample were also acquired each time the grafts wereimaged to determine blood radioactivity (blood standard). Regions ofinterest of the graft and expansion segments were selected to determinethe deposited and circulation radio-activity in the dynamic image. Thetotal number of platelets deposited on the vascular graft material andin the expansion chamber were calculated.

In 6 animals treated with CHO-rec-KRIX-1E, platelet deposition was lowerthan in the control animal treated with saline in both the venous andarterial thrombosis chambers (FIG. 26).

Example 20 Treatment of Sepsis Related Conditions with InhibitoryAntibodies Against Factor VIII with Modified Glycosylation

Injection of endotoxin elicits the production of pro-inflammatorycytokines among which IL-6 and TNF-α are important for theirinteractions with the coagulation system. Thus, IL-6 increases theproduction of tissue Factor and, consequently, the generation ofthrombin. It also increases the production of fibrinogen by anindependent mechanism. TNF-α increases the levels of plasminogenactivation inhibitor type I (PAI-1) and thereby reduces fibrinolysis.

Groups of six mice (C57B1/6) are constituted for each treatment.

Wild type and F VIII knock-out mice are intravenously injected with 30and 100 microgram of the following antibodies:

no antibody

control antibody (IgG4)

mutated Krix 1 at Asn47 (CHO-recKRIX-1Q)

(KRIX-1)/(CHO-recKRIX-1Q) in a ¼ or other ratio

Other mixtures comprising KRIX-1 or KRIX-1 derivatives are envisaged fortesting such as mixtures comprising fragments of native ordeglycosylated KRIX-1 (more particularly, Fab or scFv fragments). Othermixtures comprising KRIX-1 or KRIX-1 derivatives and a second antibody(as disclosed in example 13) or derivatives thereof are also considered.A particular mixture comprises KRIX-1 and RHD5 or fragments of KRIX-1and/or RHD5.

60 minutes after the administration of the antibody, the different mousepopulation are injected intraperitoneously with either microgram4microgram or 40 microgram or 400 microgram lipopolysaccharide (from E.coli serotype 0:111:B4) per 20 g of body weight. 90 minutes later, foreach experimental setting blood is taken of part of the population bycardiac puncture in citrate buffer for evaluation of cytokine andcoagulation factor levels. Plasma is obtained by centrifugation for 5minutes at 5,000 rpm.

The survival of the remaining mice is followed for one week.

The extent to which the fibrinolytic pathway is by a lipopolysaccharideinjection of 40 microgram per 20 g body weight is evaluated by measuringconcentrations of the two main pathway inactivators, namely PAI-1(Plasminogen activator inhibitor-I) and α₂-antiplasmin, using asandwich-type ELISA with two specific monoclonal antibodies directedtowards different sites of the molecule under evaluation.

The evolution of fibrinogen plasma concentrations is used as a readingof its conversion into fibrin.

Determination of zymogen and activated protein C can be measured forexample in accordance to Richards et al. (1990) Clin. Chem. 36,1892-1896.

The present experiment allows the identification of a suitable antibodyor mixture of antibodies in order to prevent the endotoxin relatedsepsis. Analogous experiments can be devised for other components, orconditions which lead to the upregulation of the inflammatory cytokinesIL-6 and/or TNF-alpha.

Exmaple 21 Production of Antigen Binding Fragment (Fab) of Native andDeglycosylated KRIX-1.

LCL- and CHO-KRIX-1 (0.5 mg/ml in PBS) was mixed with N-glycosidase-F(Roche Diagnostics Gmbh, Mannheim, Germany) at final concentration of 2U/ml. The mixture was incubated at 37° C. during 72 hours under gentlestirring.

Fab fragments were produced by incubating LCL- and CHO-KRIX-1 (0.5mg/ml) in phosphate buffer (KH₂PO₄ 0.039M, Na₂HPO₄ 0.068M, pH 7.0 withCysteine (0.05 M), EDTA (1 mM) and papain (10 microgram/ml). After 3 hincubation at 37° C., the reaction was stopped by adding 0.075MIodoacetamine. After 30 min at 20° C., the mixture was dialysed againstphosphate buffered saline (PBS). Undigested antibodies were removed byadsorption on HiTrap Protein A (Pharmacia).

The inhibitory activity of native and deglycosylated KRIX-1 Fab wasassessed in a Bethesda assay (Kasper et al. (1975), cited supra) and isshown in FIG. 28.

Example 22 Production and Characterization of KRIX-1 and KRIX-1Q scFvFragment

Cloning of scFv-KRIX-1VLVH in Pichia Expression Vector

An scFv fragment of KRIX-1 was constructed by adding a linker sequencebetween the 3′ end of the KRIX-1 light chain variable part (VL) and the5′end of the heavy chain variable part (VH). This was obtained by PCRamplification of KRIX-1 light chain and heavy chains using the followingprimers:

For the light chain: forward primer5′-gtatctctcgagaaaagaGAAATTGTGT-TGACGCAGTCTCCAGGC-3′ [SEQ ID NO:17]corresponding to the 5′ end of the KRIX-1 VL sequence (capital), andcontaining a XhoI restriction site (underlined) and a KEX1 sequence(bold italic); reverse primer5′-cgccagagccacctccgcctgaaccgcctccacc-TCGTTTGATCTCCACCTTGGTC [SEQ IDNO:18] corresponding to the 3′ end of the KRIX-1 Jk sequence (capital),and containing a part of the linker sequence (italic)

For the heavy chain: forward primer5′-caggcggaggtggctctggcggtg-gcggatcgCAGGTMCAGCTGGTGCAGTCTGGG-3′ (SEQ IDNO:19) corresponding to the 5′ end of the KRIX-1 VH sequence (capital),and containing a part of the linker sequence (italic); reverse primer5′-gatctctagaTGAGGAGACGGTGACCAGGGTTCC [SEQ ID NO:20] corresponding tothe 3′ end of the KRIX-1 JH sequence (capitals), and containing a XbaIrestriction site (underlined)

The PCR products were annealed and a second PCR was performed using theforward primer for the light chain (SEQ ID NO:17) and the reverse primerfor the heavy chain (SEQ ID NO: 20). The resulting scFv-KRIX-1VLVH wascloned into the pPICZalphaC expression vector (Invitrogen, Merelbeke,Belgium)

Cloning of scFv-KRIX-1VLVH with His(6)tag in Pichia Expression Vector

A SalI restriction site was added to the scFv-KRIX-1VLVH sequence inorder to clone it in frame with the His(6) sequence included in thepPICZalphaC expression vector (Invitrogen; Merelbeke; Belgium). This wasobtained by PCR using the forward primer5′-gtatctctcgagaaaagaGAAATTGTGTTGACGCAGTCTCCAGGC-3′ (SEQ ID NO:21)corresponding to the 5′ end of the KRIX-1 VL sequence (capital), andcontaining a XhoI restriction site (underlined) and a KEX1 sequence(bold italic); and the reverse primer5′-catggtcgacTGAGGAGACGGTGACCAGGGTTCCCCGGCC-3′ (SEQ ID NO:22)corresponding to the 3′ end of the KRIX-1 heavy chain JH sequence(capital), and containing a SalI restriction site (underlined).

The final pPICZalphaC-scFv-KRIX-1VLVH(His) vector was used to transformX33 cells for scFv production. The supernatant was tested to demonstratethe presence of a functional scFv fragment.

The scFv fragment was purified using the HisTrap Kit (Amersham PharmaciaBiotech, Uppsala, Sweden). After concentration the scFvKRIX-1VLVH(His)was tested in a Factor VIII chromogenic assay to evaluate the ability ofthe scFvKRIX-1VLVH(His) to inhibit Factor VIII activity. The Factor VIIIinhibitory capacity was evaluated in a Besthesda assay according to themethod in example 1 and is shown in FIG. 29.

Cloning of scFv-KRIX-1VLVHQ with His(6)tag in Pichia Expression Vector

The scFv-KRIX-1VLVHQ(His) was produced by site directed mutagenesis onthe pPICZalphaC-scFv-KRIX-1VLVH(His) resulting in a single amino acidchange in the heavy chain replacing Asn47 by a glutamine in order todisrupt the N-linked glycosylation site at Asn47-Thr49

This was obtained using the Site Directed Mutagenesis Kit (Stratagene,La Jolla, Calif.) in combination with the following specific PCRprimers:

Forward primer: 5′-CCTGCAAGACCTCTGGATACcAaTTCACCGGCTAC-TCTGCTTCTGG-3′(SEQ ID NO: 23) corresponding to nt 119 to 164 of the KRIX-1 Heavy chainsequence (capital) containing two altered nucleotides (a to c and c toa; bold italic).

Reverse primer: 5′-CCAGAAGCAGAGTAGCCGGTGAAtTgGTATC-CAGAGGTCTTGCAGG-3′(SEQ ID NO: 24) corresponding to nt 119 to 164 of the KRIX-1 Heavy chainsequence (capital) containing two altered nucleotides (g to t and t tog; bold italic).

The full length nucleotide and protein sequence of scFv-KRIX-1VLVHQ withHis(6)tag is described in SEQ ID NO: 25 and 26.

Cloning of scFv-KRIX-1VLVH and scFvKRIX-1VLVHQ(His) with His(6)tag in aCHO Expression Vector

The KRIX-1 light chain leader sequence was introduced intopPICZalphaC-scFv-KRIX-1VLVH(His) and pPICZalphaC-scFv-KRIX-1VLVHQ(His)by cloning of a HindIII/PstI restriction fragment of pCR4-KRIX-1Lcontaining the leader sequence into HindIII/PstI digestedpPICZalphaC-scFv-KRIX-1VLVH and pPICZalphaC-KRIX-1VLVHQ respectively.The resulting scFv sequence was adapted for cloning and expressionpurposes by PCR using the following specific primers:

Forward primer: 5′-cccaagcttgccgccaccATGGAAACCCCAGCKCAGCTTC-3′ (SEQ IDNO:27) corresponding to the 5′ end of the KRIX-1 Light chain sequence(capital), and containing a HindIII site (underlined) and a Kozaksequence (bold italic).

Reverse primer:5′-ccggaattctcaatgatgatgatgatgatgTGAGGAGACGGTGA-CCAGGGTTCC-3′ (SEQ IDNO:28) corresponding to the 3′ end of the KRIX-1 heavy chain JH sequence(capital), and containing a EcoRI site (underlined), a stop signalsequence (bold italic) and a His(6)tag sequence (italic)

The resulting PCR products were cloned into the pGEM-T-Easy vector(Promega; Leiden, Netherlands). After sequence verification thescFvKRIX-1VLVH(His) and scFv-KRIX-1VLVHQ(His) were cloned into thepEE14.4 vector (Lonza Biologics, Portsmouth, N.H.). The resulting vectorwas linearised with SalI before transfection.

The linearised vector was used for stable transfection of CKO-K1 cellsusing the FuGENE6 transfection reagent (Roche, Brussels, Belgium)according to the manufacturer's instructions. The transfected cells werecultured in glutamine-free DMEM (JRH Biosciences, Lenexa, Kans.)supplemented with FBS 10%, GS Supplement (JRH Biosciences, Lenexa,Kans.) and 50 μM L-Methionine Sulfoximine (MSX) (Sigma-Aldrich, Bornem,Belgium) for selection.

The best producers were adapted to growth in serum-free medium (EX-CELL302 serum-free medium w/o L-Glutamine, JRH Biosciences, Lenexa,Kans.)—supplemented with GS Supplement and MSX in the respectiveconcentration—by step-wise reduction of the FBS to 0%.

The supernatants were assayed for production of scFv-KRIX-1VLVH(His) andscFv-KRIX-1VLVHQ(His) in a Factor VIII chromogenic assay as described inexample. The Factor VIII inhibitory capacity of the culture supernatantis shown in FIG. 30.

1. An isolated monoclonal anti-Factor VIII antibody wherein saidantibody is binding to an epitope of the isolated Cl domain of FactorVIII, said C1 domain comprising the residue Arg at amino acid position2150 of Factor VIII, and wherein said binding is abrogated when said Argresidue is mutated into a His residue; and inhibiting Factor VIIIactivity by at most 98%, even when said antibody is, on a molar basis,in large excess of Factor VIII; or an antigen-binding fragment of saidantibody.
 2. A pharmaceutical composition comprising an isolatedmonoclonal anti-Factor VIII antibody or fragment thereof of claim 1 inadmixture with a pharmaceutically acceptable carrier.
 3. A method oftreatment in a mammal of a thrombotic pathological condition involvingFactor VIII, wherein in said condition the risk of clot formation isincreased, said method comprising administering to said mammal in needof such treatment a therapeutically effective amount of an antibody orfragment thereof of claim
 1. 4. A method of treatment in a mammal of athrombotic pathological condition involving Factor VIII, wherein in saidcondition the risk of clot formation is increased, said methodcomprising administering to said mammal in need of such treatment atherapeutically effective amount of a composition of claim 2.